ES2843654T3 - RNA interference compositions and methods for malignant tumors - Google Patents

Abstract

Composition comprising iRNA molecules targeting a human GST-p, iRNA molecules targeting a human p21 encoded by a CDKN1A gene, and a pharmaceutically acceptable carrier.

Description

DESCRIPTION

RNA interference compositions and methods for malignant tumors

Technical field of the invention

This invention relates to the fields of biopharmaceuticals and therapeutic agents that are composed of nucleic acid-based molecules. More particularly, this invention relates to methods and compositions for administering RNA interference agents to prevent, treat, or ameliorate the effects of conditions and diseases involving malignant tumors.

Background of the invention

Mutation of a KRAS gene can be associated with malignant tumors, such as lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, and colorectal carcinoma. Recent observations indicate that elevated levels of the glutathione S-transferase-rc protein (GST-rc) are associated with such KRAS mutations.

Without wishing to be bound by any particular theory, it has been found that upon suppression of GST-rc in cells, the level of the cell cycle regulating protein p21 can be surprisingly high.

One of the functions of the p21 protein that regulates the cell cycle is to inhibit apoptosis. For example, p21 can have the effect of protecting a cell from apoptosis induced by a chemotherapeutic agent, both in vitro and in vivo. See, for example, Gartel and Tyner, 2002, Mol Cancer Ther., 2002, 1 (8): 639-49; Abbas and Dutta, 2009, Nat Rev Cancer., 2009, 9 (6): 400-14. p21 is encoded by the CDKN1A gene and belongs to the CIP / KIP family. p21 can function to inhibit cell cycle progression in the G1 phase and the G2 / M phase by binding to a cyclin-CDK complex. For example, the p21 gene is activated by p53, a tumor suppressor gene. Upon activation of p53 due to DNA damage, p53 activates p21 such that the cell cycle stops at the G1 phase and the G2 / M phase.

GST-rc is a member of the glutathione S-transferase family of six isoenzymes (IUBMB EC 2.5.1.18) that play a role in detoxification by catalyzing the conjugation of hydrophobic and electrophilic compounds with reduced glutathione. The GST-rc gene (GSTP1) is a polymorphic gene encoding active and functionally different GSTP1 variant proteins believed to function in xenobiotic metabolism. GSTP1 may play a role in cancer susceptibility and is abundantly expressed in tumor cells. See, for example, Aliya S. et al. Mol Cell Biochem. 2003 Nov; 253 (1-2): 319-327. Glutathione S-transferase- ^ is an enzyme that in humans is encoded by the GSTP1 gene. See, for example, Bora PS et al. (October 1991) J. Biol. Chem., 266 (25): 16774-16777. The GST-rc isoenzyme has been shown to catalyze the conjugation of GSH with some alkylating antineoplastics, suggesting that overexpression of GST-rc would result in tumor cell resistance.

Elevated serum levels of rc-GST have been observed in patients with various gastrointestinal malignancies, including stomach, esophageal, colon, pancreatic, hepatocellular, and bile duct cancers. More than 80% of patients with stage III or IV stomach cancer and even about 50% of those with stage I and II had elevated serum GST-rc levels. See, for example, Niitsu Y, et al. Cancer 1989 Jan 15; 63 (2): 317-23. GST-rc was found to be a useful marker for predicting tumor recurrence in mouth cancer patients after chemotherapy. See, for example, Hirata S. et al. Cancer 1992 Nov 15: 70 (10): 2381-7.

In human colorectal cancer, the KRAS mutation appears to induce GST-rc overexpression by activating AP-1. See, for example, Miyanishi et al., Gastroenterology, 2001; 121 (4): 865-74.

GST-rc expression increases in various cancer cells, which may be related to resistance to some antineoplastic drugs. See, for example, Ban et al., Cancer Res., 1996, 56 (15): 3577-82; Nakajima et al., J Pharmacol Exp Ther., 2003, 306 (3): 861-9.

Agents for suppressing GST-rc have been reported to induce apoptosis in cells. However, such compositions and techniques also caused autophagy and required the combined action of various agents. See, for example, US 2014/0315975 A1. Furthermore, GST-rc suppression has not been found to decrease or shrink tumors. For example, in a cancer that overexpressed GST-rc, tumor weights were unaffected by GST-rc suppression, although other effects were observed. See, for example, Hokaiwado et al., Carcinogenesis, 2008, 29 (6): 1134-1138.

There is an urgent need for methods and compositions to develop therapies for patients with malignancies, such as siRNA sequences, compounds and structures for the inhibition of GST-rc expression and What is needed are methods and compositions for preventing or treating malignant tumors. There is a continuing need for iRNA molecules and other structures and compositions to prevent, treat, or reduce malignant tumors.

Brief summary

This invention provides compositions and methods for human rc-GST-targeting iRNA molecules, in combination with human p21-targeting iRNA molecules encoded by the CDKN1A gene. The compositions include a pharmaceutically acceptable carrier. Human p21 according to the invention as described below is encoded by the CDKN1A gene.

This invention relates to molecules and compositions thereof for use in biopharmaceuticals and therapeutic agents for malignant tumors. More particularly, this invention relates to compounds, compositions, and methods for providing nanoparticles for delivering and delivering active agents or pharmacological compounds to cells, tissues, organs, and subjects having malignant tumors.

Compositions according to the invention are included for use in methods of preventing, treating or ameliorating one or more symptoms of a malignant tumor in a subject in need thereof. The method may involve administering to the subject an effective amount of a composition of GST-rc and p21 targeting iRNA molecules.

Embodiments of this invention include the following:

a composition comprising human GST-rc-targeted iRNA molecules, human p21-targeted iRNA molecules, and a pharmaceutically acceptable carrier. RNAi molecules can contain a 2'-deoxynucleotide at one or more of positions 2 to 8 from the 3 'end of the antisense strand.

The carrier can include liposomal nanoparticles that encapsulate the iRNA molecules. Liposomal nanoparticles can encapsulate iRNA molecules and retain at least 80% of encapsulated iRNA molecules after 1 hour of exposure to human serum. Liposomal nanoparticles can be 10 to 1000 nm in size, or 10 to 150 nm in size.

The composition can be active in treating a malignant tumor, which can be located in any organ or tissue, including lung, colon, kidney, pancreas, liver, bone, skin or intestine.

Liposomal nanoparticles can be composed of an ionizable lipid, a structural lipid, one or more stabilizing lipids, and a lipid to reduce the immunogenicity of the nanoparticles. The ionizable lipid can be selected from the group of compound 81, compound 71, compound 57, compound 84, compound 49, compound 76, compound 78 and compound 102.

This invention further contemplates compositions according to the invention for use in methods of preventing, treating or ameliorating one or more symptoms of a malignant tumor in a subject in need thereof. The method may involve administering to the subject an effective amount of an above composition.

In some embodiments, the malignant tumor may be associated with a KRAS mutation, the method further comprising identifying a tumor cell in the subject, the tumor cell comprising at least one of: (i) a KRAS gene mutation, and (ii) a level of aberrant expression of KRAS protein.

In certain embodiments, the malignant tumor can overexpress GST-rc.

IRNA molecules can decrease the expression of GST-rc and p21 in the subject. In some embodiments, administration can decrease the expression of GST-rc and p21 in the subject by at least 5% for at least 5 days. Administration can decrease the volume of the malignant tumor in the subject by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%. The method can reduce one or more symptoms of the malignant tumor, or delay or terminate the progression of the malignant tumor.

In certain embodiments, administration can reduce the growth of malignant tumor cells in the subject. Administration can reduce growth to at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20% of the malignant tumor cells in the subject.

In some aspects, the malignant tumor may be colon cancer, pancreatic cancer, kidney cancer, lung cancer, breast cancer, fibrosarcoma, lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, or colorectal carcinoma.

Embodiments of this invention can provide compositions according to the invention for use in methods where administration is performed from 1 to 12 times per day. Administration can be carried out for a duration of 1, 2, 3, 4, 5, 6 or 7 days, or for a duration of 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks.

In some embodiments, the administration can be a dose of from 0.01 to 2 mg / kg of the iRNA molecules at least once per day for a period of up to twelve weeks. In additional embodiments, administration can provide a mean (0-ultimate) AUC of from 1 to 1000 ug * min / ml and a mean Cmax of from 0.1 to 50 ug / ml for the GST-rc iRNA molecule. In certain embodiments, administration can provide a mean (0-last) AUC of from 1 to 1000 ug * min / ml and a mean Cmax of from 0.1 to 50 ug / ml for the p21 iRNA molecule.

Methods of administration can be intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, oral, topical, infusion or inhalation.

Brief description of the drawings

Figure 1: Figure 1 shows a profound reduction of cancer xenograft tumors in vivo using a siRNA formulation of GST-rc and p21 of this invention. The GST-rc and p21 siRNAs provided gene attenuation potency in vivo when administered in a liposomal formulation to cancer xenograft tumors. A cancer xenograft model was used with a relatively low dose at 1.15 mg / kg for the GST-rc siRNA and 0.74 mg / kg for the p21 siRNA. The siRNA formulation of GST-rc and p21 showed significant tumor inhibition efficacy within a few days after administration. After 30 days, the siRNAs of GST-rc and p21 showed a markedly advantageous tumor inhibition efficiency, with a tumor volume reduced by more than 2-fold compared to the control.

Figure 2: Figure 2 shows a profound reduction of cancer xenograft tumors in vivo using a siRNA formulation of GST-rc and p21 of this invention. The GST-rc and p21 siRNAs provided gene attenuation potency in vivo when administered in a liposomal formulation to cancer xenograft tumors. A cancer xenograft model was used with a relatively low dose at 0.75 mg / kg for each siRNA. The siRNA formulation of GST-rc and p21 showed significant tumor inhibition efficacy within a few days after administration. After 30 days, the GST-rc and p21 siRNAs showed markedly advantageous tumor inhibition efficiency, with a 1.7-fold reduced tumor volume compared to the control.

Figure 3: Figure 3 shows that the siRNA formulations of GST-rc and p21 of this invention demonstrated cancer cell death by cancer cell apoptosis in vitro. Cancer cell apoptosis was monitored in vitro by observing up-regulation of PUMA, a biomarker for apoptosis, which is associated with loss of cell viability. As shown in Figure 3, the expression level of PUMA for a siRNA formulation of GST-rc and p21 was greatly increased from about 2-4 days from transfection of the GST-rc and p21 siRNAs.

Figure 4: Figure 4 shows deep reduction of orthotopic lung cancer tumors in vivo by a siRNA of this invention targeting GST-rc. GST-rc siRNA was administered in a liposomal formulation at a dose of 2 mg / kg to athymic nude mice bearing A549 orthotopic lung cancer tumors. Final weights of primary tumors were measured at necropsy for the treatment group and a vehicle control group. The GST-rc siRNA showed significant efficacy in inhibiting lung cancer tumors in this six-week study. As shown in Figure 4, after 43 days, the rc-GST siRNA showed markedly advantageous tumor inhibition, with final average inmates of primary tumors significantly reduced by 2.8-fold, compared to control.

Figure 5: Figure 5 shows tumor inhibition efficiency in vivo for a GST-rc siRNA. A cancer xenograft model was used using A549 cells with a relatively low dose of siRNA at 0.75 mg / kg. The GST-rc siRNA showed advantageous tumor inhibition within a few days. After 36 days, the rc-GST siRNA showed markedly advantageous tumor inhibition, with final average tumor volumes significantly reduced by approximately 2-fold, compared to the control.

Figure 6: Figure 6 shows that a GST-rc siRNA of this invention greatly increased cancer cell death by apoptosis in vitro. The GST-rc siRNA elicited up-regulation of PUMA, a biomarker for apoptosis, which is associated with loss of cell viability. In Figure 6, PUMA expression was greatly increased from 2-6 days after transfection of the GST-rc siRNA.

Figure 7: Figure 7 shows that a GST-rc siRNA of this invention provided attenuation efficiency for A549 xenograft tumors in vivo. Dose-dependent attenuation of GST-rc mRNA was observed in nude athymic female (nu / nu) mice (Charles River) with siRNA targeting GST-rc. As shown in Figure 7, at a dose of 4 mg / kg, a significant reduction of approximately 40% in GST-rc mRNA was detected 24 hours after injection.

Detailed description of the invention

This disclosure provides compounds and compositions for use in therapeutic combinations for administration to malignant tumors. In some aspects, this disclosure relates to compounds, compositions, and methods for providing nanoparticles for delivering and delivering active agents or pharmacological compounds to malignant tumor cells, as well as to tissues, organs, and subjects having malignant tumors.

This disclosure provides a range of ionizable compounds for delivering the active agents to malignant tumor cells. Among other uses, the ionizable compounds of this disclosure can be used to form nanoparticles to deliver and deliver active agents to enhance malignant tumors.

Aspects of this disclosure include a wide range of compounds that have lipid-like properties, and such compounds can be used to deliver active agents for uptake into malignant tumor cells.

The compositions and methods of this disclosure can be used to deliver agents to suppress gene expression. Examples of an agent for suppressing gene expression include inhibitory nucleic acid molecules, including ribozymes, antisense nucleic acids, and RNA interference molecules (iRNA molecules).

In another aspect, this disclosure provides methods of using therapeutic compositions that decrease the expression of a GST-rc nucleic acid or polypeptide molecule and a p21 nucleic acid or polypeptide molecule for the treatment of a neoplasm in a subject, wherein the neoplasm it is associated with cells that contain a KRAS mutation or that show aberrant KRAS expression levels.

Therapeutic compositions of this disclosure may include inhibitory nucleic acid molecules such as siRNA, shRNA, and antisense RNA, as well as DNA-directed RNA (ddRNA), Piwi-interacting RNA (piRNA), and repeat-associated siRNA (rasiRNA).

A KRAS-associated malignant tumor or KRAS-associated cancer is defined herein as (a) a cancer cell or a tumor cell containing a somatic KRAS mutation, or (b) a cancer cell or a tumor cell with a level of abnormal KRAS expression including, but not limited to, amplification of the DNA encoding KRAS, or overexpression of the KRAS gene, or under-expression of the KRAS gene compared to the level found in normal non-cancer cells.

GST-rc indicates an enzyme, which is encoded by the GSTP1 gene and catalyzes the conjugation of glutathione. GST-rc is present in various animals, including humans, and its sequence information is known and provided in the NCBI database accession numbers (for example, human: NP_000843 (NM_000852), rat: NP_036709 ( NM_012577), mouse: NP_038569 (NM_013541), etc.

This disclosure encompasses iRNA molecules for deleting DNA encoding GST-rc, ribozymes, antisense nucleic acids, DNA / RNA chimeric polynucleotides and vectors to express them, and GST-rc dominant negative variants.

p21 is present in various animals, including humans, and its sequence information is also publicly known (for example, human: NM_000389.4, NM_078467.2, NM 001291549.1, NM 001220778.1, NM 001220777.1 (NP_001207707.1, NP_001278478.1 , NP_001207706.1, NP_510867.1, NP_000380.1), etc .; the numbers represent the NCBI database record numbers, and the nucleotide sequence and amino acid sequence are indicated outside and inside the parentheses, respectively). As an example, the nucleotide sequence of the human CDKN1A gene is registered in the database as NM_000389.4. As for p21, the sequence information has been registered with a plurality of registration numbers as mentioned above, and a plurality of transcript variants are present.

This disclosure encompasses iRNA molecules for deleting DNA encoding p21, ribozymes, ^{antisense nucleic acids, chimeric} DNA / RNA ^{polynucleotides and vectors to express them, and dominant negative variants} of p21.

In general, after a subject is diagnosed as having a neoplasm, eg, lung cancer, kidney cancer, or pancreatic cancer, associated with a KRAS mutation or KRAS amplification, a treatment method involving the deletion of GST-rc and p21.

Examples of a GST-rc suppressing agent as used herein include a drug that suppresses GST-rc production and / or activity, and a drug that promotes GST-rc degradation and / or inactivation. Examples of the drug that suppresses GST-rc production include an iRNA molecule, a ribozyme, an antisense nucleic acid, a DNA / RNA chimeric polynucleotide for the DNA encoding GST-rc or a vector expressing the same.

Examples of an agent that suppresses p21 as used herein include a drug that suppresses the production and / or activity of p21, and a drug that promotes the degradation and / or inactivation of p21. Examples of the drug that suppresses p21 production include an iaRn molecule, a ribozyme, an antisense nucleic acid, a chimeric DNA / RNA polynucleotide for the DNA encoding p21 or a vector expressing the same.

IRNA molecules

One of skill in the art would understand that a reported sequence can change over time and incorporate any necessary changes in nucleic acid molecules herein accordingly.

Aspects of this disclosure can provide compositions and methods for gene attenuation of GST-rc expression using nucleic acid molecules.

Aspects of this disclosure can provide compositions and methods for gene silencing of p21 expression using nucleic acid molecules.

Aspects of this disclosure can provide compositions and methods for gene silencing of a combination of GST-rc and p21 expression using nucleic acid molecules.

Examples of nucleic acid molecules capable of mediating RNA interference include molecules active in RNA interference (iRNA molecules), including a duplex RNA such as siRNA ( ^{small interfering RNA), miRNA (micro} aRn), ^{shRNA (Short hairpin RNA), ddRNA (RNA directed by} DNA), ^{piRNA (RNA that} interacts with Piwi) or rasiRNA (siRNA associated with repeats) and modified forms thereof.

The composition and methods disclosed herein can also be used in the treatment of various classes of malignant tumors in a subject.

The nucleic acid molecules and methods of this disclosure can be combined, or used in combination to down-regulate the expression of genes encoding GST-rc, and to down-regulate the expression of genes encoding p21.

The compositions and methods of this disclosure can include one or more nucleic acid molecules, which in combination can modulate or regulate the expression of the GST-rc and p21 proteins and / or genes that encode the proteins, proteins and / or genes. encoding proteins that are associated with the maintenance and / or development of diseases, as well as conditions or disorders associated with GST-rc and p21, such as malignancy. The compositions and methods of this disclosure are described with reference to exemplary sequences of GST-rc and p21. One of ordinary skill in the art would understand that various aspects of the disclosure refer to any related GST-rc or p21 gene, sequence or variant, such as homologous genes and transcript variants, and polymorphisms, including single nucleotide polymorphism (SNP ) associated with any GST-rc or p21 gene. In some aspects, the compositions and methods of this disclosure can provide a double-stranded short interfering nucleic acid (siRNA) molecule that down-regulates the expression of a GST-rc gene, eg, human GST-rc. The compositions and methods of this disclosure further contemplate providing a double-stranded short interfering nucleic acid (siRNA) molecule that down-regulates the expression of a p21 gene, eg, human p21.

An iRNA molecule of this disclosure can be targeted to GST-rc or p21, and any homologous sequence, for example, using complementary sequences or incorporating non-canonical base pairs, for example mismatches and / or rocking base pairs, which can provide additional target sequences. In cases where mismatches are identified, non-canonical base pairs, eg, mismatches and / or rocking bases, can be used to generate nucleic acid molecules targeting more than one gene sequence.

For example, non-canonical base pairs such as the UU and CC base pairs can be used to generate nucleic acid molecules that are capable of targeting sequences for different targets that share sequence homology. Thus, an iRNA molecule can target a nucleotide sequence that is conserved among homologous genes, and a single iRNA molecule can be used to inhibit the expression of more than one gene.

In some aspects, the compositions and methods of this disclosure include iRNA molecules that are active against any portion of GST-rc mRNA. The iRNA molecule can include a sequence complementary to any mRNA that encodes a GST-rc sequence. This disclosure also contemplates compositions and methods that include iRNA molecules that are active against any portion of the p21 mRNA. The iRNA molecule can include a sequence complementary to any mRNA that encodes a p21 sequence.

In some aspects, an iRNA molecule of this disclosure may have activity against GST-rc RNA, wherein the iRNA molecule includes a sequence complementary to an RNA having a variant GST-rc coding sequence, for example, a mutant GST-rc gene known in the art to associate with a malignant tumor. In some embodiments, an iRNA molecule of this disclosure may have activity against p21 RNA, wherein the iRNA molecule includes a sequence complementary to an RNA having a variant p21 coding sequence, eg, a mutant p21 gene. known in the art to be associated with malignancy.

In additional aspects, an iRNA molecule of this disclosure can include a nucleotide sequence that can mediate the silencing of the expression of the GST-rc gene. In additional aspects, an iRNA molecule of this disclosure can include a nucleotide sequence that can mediate the silencing of the expression of the p21 gene.

Examples of iRNA molecules comprised in compositions of this invention, as defined in the claims, directed to GST-rc mRNA are shown in Table 1.

Table 1: sequences of iRNA molecules for GST-rc

Legend for Table 1: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lower case letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine, respectively.

Examples of iRNA molecules comprised in compositions of this invention, as defined in the claims, targeting GST-rc mRNA are shown in Table 2.

Table 2: sequences of iRNA molecules for GST-rc

Legend for Table 2: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t ) respectively. Underlining refers to substituted with 2'-OMe, eg U. Lowercase letter f refers to substitution with 2'-deoxy-2'-fluoro, eg fU is 2'-deoxy-2'- fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or a modified, inverted, or chemically modified nucleotide.

Examples of iRNA molecules comprised in compositions of this invention, as defined in the claims, targeting GST-rc mRNA are shown in Table 3.

Table 3: sequences of iRNA molecules for GST-rc

Key to Table 3: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t ) respectively. Underlining refers to substituted with 2'-OMe, eg U. Lowercase letter f refers to substitution with 2'-deoxy-2'-fluoro, eg fU is 2'-deoxy-2'- fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or a modified, inverted, or chemically modified nucleotide.

Examples of iRNA molecules comprised in compositions of this invention, as defined in the claims, targeting GST-rc mRNA are shown in Table 4.

Table 4: sequences of iRNA molecules for GST-rc

Key to Table 4: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t ) respectively. Underlining refers to substituted with 2'-OMe, eg U. Lowercase letter f refers to substitution with 2'-deoxy-2'-fluoro, eg fU is 2'-deoxy-2'- fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or a modified, inverted, or chemically modified nucleotide.

Examples of iRNA molecules comprised in compositions of this invention, as defined in the claims, directed to GST-rc mRNA are shown in Table 5.

Table 5: sequences of iRNA molecules for GST-rc

Key to Table 5: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t ) respectively. Underlining refers to substituted with 2'-OMe, eg U. Lowercase letter f refers to substitution with 2'-deoxy-2'-fluoro, eg fU is 2'-deoxy-2'- fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or a modified, inverted, or chemically modified nucleotide.

Examples of iRNA molecules comprised in compositions of this invention, as defined in the claims, targeting GST-rc mRNA are shown in Table 6.

Table 6: sequences of iRNA molecules for GST-rc

Legend for Table 6: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t ) respectively. Underlining refers to substituted with 2'-OMe, eg U. Lowercase letter f refers to substitution with 2'-deoxy-2'-fluoro, eg fU is 2'-deoxy-2'- fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or a modified, inverted, or chemically modified nucleotide.

Examples of iRNA molecules comprised in compositions of this invention, as defined in the claims, targeting p21 mRNA are shown in Table 7.

Table 7: sequences of iRNA molecules for p21

Legend for Table 7: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lower case letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C and deoxythymidine respectively. mU is 2'-methoxy-U.

Examples of iRNA molecules comprised in compositions of this invention, as defined in the claims, targeting p21 mRNA are shown in Table 8.

Table 8: sequences of iRNA molecules for p21

Legend for Table 8: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, and deoxythymidine (dT = T = t ) respectively. Underlining refers to substituted with 2'-OMe, eg, U. N is A, C, G, U, U, a, c, g, u, t, or a modified, inverted, or chemically modified nucleotide.

In some aspects, this disclosure provides a range of nucleic acid molecules, wherein a) the molecule has a sense polynucleotide strand and an antisense polynucleotide strand; b) each strand of the molecule is from 15 to 30 nucleotides in length; c) a contiguous region of from 15 to 30 nucleotides of the antisense strand is complementary to a sequence of an mRNA encoding p21; and d) at least a portion of the sense strand is complementary to at least a portion of the antisense strand, and the molecule has a duplex region of from 15 to 30 nucleotides in length.

In some aspects, the nucleic acid molecule may have a contiguous 15 to 30 nucleotide region of the antisense strand that is complementary to a sequence of an mRNA encoding p21, and is located in the duplex region of the molecule.

In additional aspects, the nucleic acid molecule may have a contiguous 15 to 30 nucleotide region of the antisense strand that is complementary to a sequence of an mRNA encoding p21.

In additional aspects, a nucleic acid molecule of this disclosure can have each strand of the molecule that is from 18 to 22 nucleotides in length. A nucleic acid molecule can have a duplex region 19 nucleotides in length.

In certain aspects, a nucleic acid molecule may have a polynucleotide sense strand and the polynucleotide antisense strand that are connected as a single strand, and form a duplex region connected at one end by a loop.

The nucleic acid molecules of this disclosure may have a blunt end, and may have one or more 3 'projections.

The nucleic acid molecules of this invention are iRNA molecules that are active for gene silencing, for example, a dsRNA that is active for gene silencing, a siRNA, a micro-RNA, or an shRNA that is active for gene silencing, as well as a RNA directed by DNA (ddRNA), an RNA that interacts with Piwi (piRNA), and a siRNA associated with repeats (rasiRNA).

This disclosure provides a range of nucleic acid molecules that are active in inhibiting the expression of p21. In some aspects, the nucleic acid molecule may have an IC50 for p21 attenuation of less than 100 pM.

In additional aspects, the nucleic acid molecule may have an IC50 for p21 attenuation of less than 50 pM.

This disclosure further contemplates compositions containing one or more nucleic acid molecules of the invention and a pharmaceutically acceptable carrier. The carrier can be a lipid molecule or liposome.

The compounds and compositions of this disclosure are useful in methods of preventing or treating a disease associated with p21, administering a compound or composition to a subject in need thereof.

In additional aspects, this disclosure includes methods for treating a disease associated with the expression of p21, by administering to a subject in need thereof a composition containing one or more nucleic acid molecules of the invention. The disease can be a malignant tumor, which can present in a disease such as cancers associated with the expression of p21, among others.

In some aspects, this disclosure provides a range of nucleic acid molecules, wherein: a) the molecule has a sense polynucleotide strand and an antisense polynucleotide strand; b) each strand of the molecule is from 15 to 30 nucleotides in length; c) a contiguous region of from 15 to 30 nucleotides of the antisense strand is complementary to a sequence of an mRNA encoding GST-rc; d) at least a portion of the sense strand is complementary to at least one portion of the antisense strand, and the molecule has a duplex region of 15 to 30 nucleotides in length.

In some aspects, the nucleic acid molecule may have a contiguous 15 to 30 nucleotide region of the antisense strand that is complementary to a sequence of an mRNA encoding GST-rc that is located in the duplex region of the molecule.

In additional aspects, the nucleic acid molecule may have a contiguous 15 to 30 nucleotide region of the antisense strand that is complementary to a sequence of an mRNA encoding GST-rc. In certain aspects, each strand of the nucleic acid molecule can be from 18 to 22 nucleotides in length. The duplex region of nucleic acid molecules can be 19 nucleotides in length. In alternative forms, the nucleic acid molecule may have a sense polynucleotide strand and an antisense polynucleotide strand that are connected as a single strand, and form a duplex region connected at one end by a loop.

Some aspects of a nucleic acid molecule of this disclosure may have a blunt end. In certain aspects, a nucleic acid molecule can have one or more 3 'projections.

This disclosure provides a range of nucleic acid molecules that are active iRNA molecules for gene silencing. The nucleic acid molecules of the invention can be a dsRNA, a siRNA, a micro-RNA, or an active shRNA for gene silencing, as well as a DNA-directed RNA (ddRNA), RNA that interacts with Piwi (piRNA), or SiRNA associated with repeats (rasiRNA). Nucleic acid molecules can be active in inhibiting GST-rc expression.

Aspects of this disclosure further provide nucleic acid molecules having an IC50 for GST-rc attenuation of less than 100 pM.

Additional aspects of this disclosure provide nucleic acid molecules having an IC50 for GST-rc attenuation of less than 50 pM.

This disclosure further contemplates compositions containing one or more of the nucleic acid molecules of the invention, together with a pharmaceutically acceptable carrier. In certain aspects, the carrier can be a lipid molecule or liposome.

The compounds and compositions of this disclosure are useful in methods of preventing or treating a disease associated with rc-GST by administering a compound or composition to a subject in need thereof.

As used herein, the iRNA molecule denotes any molecule that causes RNA interference, including a duplex RNA such as siRNA (small interfering RNA), miRNA (micro RNA), shRNA ^{(short hairpin RNA), ddRNA (RNA-directed} RNA), ^{piRNA (RNA that interacts with Piwi), or rasiRNA (siRNA} associated with repeats) and modified forms thereof. These iRNA molecules can be commercially available or can be designed and prepared based on known sequence information, etc. Nucleic acid includes RNA, DNA, PNA, or a complex thereof. As used herein, the DNA / RNA chimeric polynucleotide includes a double-stranded polynucleotide that is composed of DNA and RNA that inhibits the expression of a target gene.

In one aspect, the agents of this disclosure contain siRNA as a therapeutic agent. A siRNA molecule can be from about 10-50 or more nucleotides in length. A siRNA molecule can be from about 15-45 nucleotides in length. A siRNA molecule can be from about 19-40 nucleotides in length. A siRNA molecule can be from 19-23 nucleotides in length. A siRNA molecule of this invention can mediate iRNAs against a target mRNA. Commercially available design tools and kits, such as those available from Ambion, Inc. (Austin, TX), and the Whitehead Institute of Biomedical Research at MIT (Cambridge, MA) allow the design and production of siRNAs.

Methods for treating a malignant tumor

Embodiments of this invention can provide compositions comprising a pharmaceutically acceptable carrier and iRNA molecules that can be used to down-regulate or inhibit the expression of GST-n and / or GST-n proteins, as well as iRNA molecules that can be used to down-regulate the expression of p21 and / or p21 proteins.

In some embodiments, an iRNA molecule comprised in compositions of this invention, as defined in the claims, can be used to down-regulate or inhibit the expression of GST-n and / or GST-n proteins arising from haplotype polymorphisms. GST-n that can be associated with a disease or a condition such as a malignant tumor. The iRNA molecules comprised in compositions of this invention, as defined in the claims, can be used to down-regulate or inhibit the expression of p21 and / or p21 proteins arising from p21 haplotype polymorphisms that may be associated with disease or a state such as a malignant tumor.

Monitoring of GST-n mRNA or protein levels, as well as p21 mRNA or protein levels, can be used to characterize gene silencing and to determine the efficacy of the compounds and compositions of this invention.

The iRNA molecules of this disclosure can be used individually or in combination with other siRNAs to modulate the expression of one or more genes.

The iRNA molecules of this disclosure can be used individually or in combination, or in conjunction with other known drugs to prevent or treat diseases, or ameliorate symptoms of conditions or disorders associated with GST-n and p21, including a malignant tumor.

The iRNA molecules comprised in compositions of this invention, as defined in the claims, can be used to modulate or inhibit GST-n expression in a sequence-specific manner. Furthermore, the iRNA molecules comprised in compositions of this invention, as defined in the claims, can be used to modulate or inhibit the expression of p21 in a sequence-specific manner. The iRNA molecules of this disclosure may include a leader strand for which a series of contiguous nucleotides are at least partially complementary to a GST-n mRNA or a p21 mRNA.

In certain aspects, the malignant tumor can be treated by RNA interference using a composition, as defined in the claims, of this invention comprising iRNA molecules, as defined in the claims.

Treatment of the malignant tumor can be characterized in suitable cell-based models, as well as in ex vivo or in vivo animal models.

The treatment of a malignant tumor can be characterized by determining the level of GST-n mRNA or the level of GST-n protein in the cells of the affected tissue. The treatment of a malignant tumor can also be characterized by determining the level of p21 mRNA or the level of p21 protein in the cells of the affected tissue.

Treatment of a malignant tumor can be characterized by a non-invasive medical examination of an affected organ or tissue.

Aspects of this disclosure may include methods for preventing, treating, or ameliorating the symptoms of a disease or condition associated with GST-n and / or p21 in a subject in need thereof.

In some aspects, methods for preventing, treating, or ameliorating the symptoms of a malignant tumor in a subject may include administering to the subject an iRNA molecule of this disclosure to modulate the expression of a GST-n gene and / or a p21 gene. in the subject or organism.

In some aspects, this disclosure contemplates methods for down-regulating the expression of a GST-n gene in a cell or organism by contacting the cell or organism with an iRNA molecule of this invention. This disclosure further contemplates methods for down-regulating the expression of a p21 gene in a cell or organism by contacting the cell or organism with an iRNA molecule of this invention.

The GST-n and p21 inhibitory nucleic acid molecules can be nucleotide oligomers that can be used as a single or double stranded nucleic acid molecule to decrease gene expression. In one approach, the inhibitory nucleic acid molecule is a double-stranded RNA used for interference-mediated RNA (iRNA) attenuation of gene expression. In one aspect, a double-stranded RNA (dsRNA) molecule is produced that is active in RNA interference that includes from eight to twenty-five (eg, 21, 22, 23, 24, 25) consecutive nucleotides of a nucleotide oligomer of the invention. DsRNA can be two strands complementary RNAs that have been duplicated, or a single strand of RNA that has been self-duplicating (short hairpin RNA (hc)).

In some aspects, dsRNAs that are active in RNA interference are about 21 or 22 base pairs, but can be shorter or longer, up to about 29 nucleotides. Double-stranded RNA can be prepared using standard techniques, eg, chemical synthesis or in vitro transcription. Kits are available, for example, from Ambion (Austin, Texas) and Epicenter (Madison, Wis.).

Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296: 550-553, 2002; Paddison et al. Genes & Devel. 16: 948-958, 2002; Paul et al. Nature Biotechnol. 20: 505-508, 2002; Sui et al., Proc. Natl. Acad. Sci. USA 99: 5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99: 6047-6052, 2002; Miyagishi et al., Nature Biotechnol. 20: 497-500, 2002; and Lee et al., Nature Biotechnol. 20: 500-5052002.

An inhibitory nucleic acid molecule that "corresponds" to a GST-rc gene comprises at least one fragment of the double-stranded gene, such that each strand of the double-stranded inhibitory nucleic acid molecule is capable of binding to the complementary strand of the GST- gene. rc diana. The inhibitory nucleic acid molecule need not have a perfect match to the reference GST-rc sequence.

An inhibitory nucleic acid molecule that "corresponds" to a p21 gene comprises at least one fragment of the double-stranded gene, such that each strand of the double-stranded inhibitory nucleic acid molecule is capable of binding to the complementary strand of the target p21 gene. The inhibitory nucleic acid molecule need not have a perfect match to the reference p21 sequence.

In one aspect, a siRNA has a sequence identity of at least about 85%, 90%, 95%, 96%, 97%, 98%, or even 99% to the target nucleic acid. For example, a 19 base pair duplex having a 1-2 base pair mismatch is considered useful in the methods of the invention. In other aspects, the nucleotide sequence of the inhibitory nucleic acid molecule exhibits 1, 2, 3, 4, 5, or more mismatches.

The inhibitory nucleic acid molecules provided by the disclosure are not limited to siRNAs, but include any nucleic acid molecule sufficient to decrease the expression of a GST-rc or p21 nucleic acid or polypeptide molecule. The DNA sequences provided herein can be used, for example, in the discovery and development of a therapeutic antisense nucleic acid molecule to decrease the expression of the encoded protein. The invention further provides catalytic RNA molecules or ribozymes. Such catalytic α-Rn molecules can be used to inhibit the expression of a target nucleic acid molecule in vivo. The inclusion of ribozyme sequences within an antisense RNA confers RNA cleavage activity on the molecule, thus increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334: 585-591. 1988 and in US 2003/0003469 A1.

In various aspects of this disclosure, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described in Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Examples of hairpin motifs are described in Hampel et al., Biochemistry, 28: 4929, 1989 and Hampel et al., Nucleic Acids Research, 18: 299, 1990. Those skilled in the art will recognize that what is needed in an enzymatic nucleic acid molecule is a specific substrate binding site that is complementary to one or more of the RNA regions of the target gene, and having nucleotide sequences within or around that substrate binding site that confer RNA cleavage activity to the molecule.

Suppression of a target can be determined by the expression or activity of the corresponding protein in cells that are suppressed, compared to cells in which a suppressing agent is not used. Protein expression can be assessed by any known technique; Examples include an immunoprecipitation method using an antibody, EIA, ELISA, IRA, IRMA, a Western blot method, an immunohistochemical method, an immunocytochemical method, a flow cytometric method, various hybridization methods that use a nucleic acid that specifically hybridizes to a nucleic acid that encodes the protein or a single fragment thereof, or a transcription product (e.g. mRNA) or a splice product of said nucleic acid, a method Northern blotting, a Southern blotting method, and various PCR methods.

The activity of the protein can be assessed by testing a known activity of the protein including binding to a protein such as, for example, Raf-1 (particularly phosphorylated Raf-1) or EGFR (particularly phosphorylated EGFR) by means of any a known method such as, for example, an immunoprecipitation method, a Western blot method, an accumulation analysis method, an extraction method or a surface plasmon resonance (SPR) method.

Examples of mutated KRAS include, but are not limited to, those that have a mutation that causes constant activation of KRAS, such as a mutation that inhibits endogenous GTPase or a mutation that increases the rate of exchange of guanine nucleotides. Specific examples of such a mutation include, but are not limited to, for example, the mutation at amino acids 12, 13 and / or 61 in human KRAS (inhibiting endogenous GTPase) and the mutation at amino acids 116 and / or 119 in Human KRAS (increasing the rate of guanine nucleotide exchange) (Bos, Cancer Res. 1989; 49 (17): 4682-9, Levi et al., Cancer Res. 1991; 51 (13): 3497-502).

In some aspects of the present disclosure, the mutated KRAS can be a KRAS that has a mutation in at least one of amino acids 12, 13, 61, 116, and 119 of human KRAS. In one aspect of the present disclosure, the mutated KRAS has a mutation at amino acid 12 of human KRAS. In some aspects, the mutated KRAS may be one that induces GST-n overexpression. Cells that have mutated KRAS can show GST-n overexpression.

Detection of mutated KRAS can be carried out using any known technique, for example, selective hybridization by means of a nucleic acid probe specific for a known mutational sequence, an enzyme mismatch cleavage method, sequencing (Bos, Cancer Res. 1989; 49 (17): 4682-9) and a PCR-RFLP method (Miyanishi et al., Gastroenterology. 2001; 121 (4): 865-74).).

Detection of target expression can be carried out using any known technique. Whether or not the target is overexpressed can be assessed by comparing, for example, the degree of expression of the target in cells having mutated KRAS with the degree of expression of the target in the same cell type having normal KRAS. In this situation, the target is overexpressed if the degree of expression of the target in cells that have mutated KRAS exceeds the degree of expression of the target in the same type of cells that have normal KRAS.

In one aspect, the disclosure features a vector that encodes a nucleic acid molecule inhibitory of any of the foregoing aspects. In a particular aspect, the vector is a retroviral, adenoviral, adeno-associated viral or lentiviral vector. In another embodiment, the vector contains a promoter suitable for expression in a mammalian cell.

The amount of active ingredient that induces RNA interference formulated in the composition of the present disclosure may be an amount that does not cause an adverse effect that exceeds the benefit of administration. Such an amount can be determined by an in vitro test using cultured cells, or a test in an animal or mammalian model, such as a mouse, rat, dog or pig, etc., and such test methods are known. by those skilled in the art. The methods of this disclosure can be applicable to any animal, including humans.

The amount of active ingredient formulated can vary depending on the form in which the agent or composition is administered. For example, when using a plurality of units of the composition for one administration, the amount of active ingredient to be formulated in one unit of the composition can be determined by dividing the amount of active ingredient necessary for one administration among said plurality of units.

This disclosure also relates to a process for producing an agent or composition to suppress GST-n and p21, and to the use of a composition that suppresses GST-n and p21 to reduce or shrink malignant tumors.

RNA interference

RNA interference (iRNA) refers to sequence-specific post-transcriptional gene silencing in animals mediated by small interfering RNA (siRNA). See, for example, Zamore et al., Cell, 2000, vol.

101, pp. 25-33; Fire et al., Nature, 1998, vol. 391, pp. 806811; Sharp, Genes & Development, 1999, vol. 13, pp.

139-141.

An RNAi response in cells can be triggered by a double-stranded RNA (dsRNA), although the mechanism is not yet fully understood. Certain dsRNAs in cells can undergo the action of the enzyme Dicer, a ribonuclease III enzyme. See, for example, Zamore et al., Cell, 2000, vol. 101, pp. 25-33; Hammond et al., Nature, 2000, vol. 404, pp. 293-296. Dicer can process dsRNA into shorter dsRNA chunks, which are siRNAs.

In general, siRNAs can be from about 21 to about 23 nucleotides in length and include a base pair duplex region of about 19 nucleotides in length.

IRNA involves an endonuclease complex known as an RNA-induced silencing complex (RISC). An siRNA has an antisense or leader strand that enters the RISC complex and mediates the cleavage of a single stranded RNA target that has a sequence complementary to the antisense strand of the siRNA duplex. The other strand of siRNA is the passing strand. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex. See, eg, Elbashir et al., Genes & Development, 2001, vol. 15, pp. 188-200.

As used herein, the term "sense strand" refers to a nucleotide sequence of a siRNA molecule that is partially or fully complementary to at least a portion of a corresponding antisense strand of the siRNA molecule. The sense strand of a siRNA molecule can include a nucleic acid sequence that has homology to a target nucleic acid sequence.

As used herein, the term "antisense strand" refers to a nucleotide sequence of a siRNA molecule that is partially or fully complementary to at least a portion of a target nucleic acid sequence. The antisense strand of a siRNA molecule can include a nucleic acid sequence that is complementary to at least a portion of a corresponding sense strand of the siRNA molecule.

IRNA molecules can down-regulate or attenuate gene expression by mediating RNA interference in a sequence-specific manner. See, for example, Zamore et al., Cell, 2000, vol. 101, pp. 25-33; Elbashir et al., Nature, 2001, vol. 411, pp. 494-498; Kreutzer et al., WO2000 / 044895; Zernicka-Goetz et al., WO2001 / 36646; Fire et al., WO1999 / 032619; Plaetinck et al., WO2000 / 01846; Mello et al., WO2001 / 029058.

As used herein, the terms "inhibit," "down-regulate," or "reduce" with respect to gene expression means that the expression of the gene, or the level of mRNA molecules that encode one or more proteins, or the activity of one or more of the encoded proteins is reduced below that observed in the absence of an iRNA or siRNA molecule of this invention. For example, the expression level, the mRNA level, or the activity level of the encoded protein can be reduced by at least 1%, or at least 10%, or at least 20%, or at least 50%. , or at least 90%, or more than observed in the absence of an iRNA or siRNA molecule of this invention.

IRNA molecules can also be used to attenuate viral gene expression and thus affect viral replication.

IRNA molecules can be prepared from separate polynucleotide strands: a sense strand or passenger strand and an antisense strand or leader strand. The guide and passing chain are at least partially complementary. The guide strand and the passing strand can form a duplex region that is from about 15 to about 49 base pairs.

In some aspects, the duplex region of an siRNA may have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs.

In certain aspects, an iRNA molecule can be active in a RISC complex, with a duplex region length active for RISC.

In additional aspects, an iRNA molecule can be active as a Dicer substrate, which is to be converted to an iRNA molecule that can be active in a RISC complex.

In some aspects, an RNAi molecule may have complementary leader and passenger sequence portions at opposite ends of a long molecule, such that the molecule may form a duplex region with the complementary sequence portions, and the strands are linked in a end of the duplex region by nucleotide or non-nucleotide linkers. For example, a hairpin arrangement or a stem and loop arrangement. The linker interactions with the chains can be covalent bonds or non-covalent interactions.

An iRNA molecule of this disclosure may include a nucleotide, non-nucleotide, or mixed nucleotide / non-nucleotide linker that links the sense region of the nucleic acid to the antisense region of the nucleic acid. A nucleotide linker can be a linker of ^ 2 nucleotides in length, eg, about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. The nucleotide linker can be a nucleic acid aptamer. By "aptamer" or "nucleic acid aptamer" as used herein refers to a nucleic acid molecule that specifically binds to a target molecule wherein the nucleic acid molecule has a sequence that includes a sequence recognized by the target molecule in its natural environment. Alternatively, an aptamer can be a nucleic acid molecule that binds to a target molecule, wherein the target molecule does not naturally bind to a nucleic acid. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thus avoiding the naturally occurring ligand interaction with the protein. See, for example, Gold et al., Annu Rev Biochem, 1995, vol. 64, pp. 763-797; Brody et al., J. Biotechnol., 2000, vol. 74, pp.

5-13; Hermann et al., Science, 2000, vol. 287, pp. 820-825.

Examples of a non-nucleotide linker include an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds, for example polyethylene glycols such as those having from 2 to 100 ethylene glycol units. Some examples are described in Seela et al., Nucleic Acids Research, 1987, vol. 15, pp. 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, vol. 113, pp. 6324-6326; Jaeschke et al., Tetrahedron Lett., 1993, vol. 34, pp. 301; Arnold et al., WO1989 / 002439; Usman et al., WO1995 / 006731; Dudycz et al., WO1995 / 011910 and Ferentz et al., J. Am. Chem. Soc., 1991, vol. 113, pp. 4000-4002.

An iRNA molecule can have one or more projections from the duplex region. The projections, which are single-stranded unpaired base regions, can be from one to eight nucleotides in length, or longer. A projection can be a 3 'end projection, in which the 3' end of a strand has a single stranded region of from one to eight nucleotides. A projection can be a 5 'end projection, in which the 5' end of a strand has a single stranded region of from one to eight nucleotides.

The projections of an iRNA molecule can be the same length, or they can be of different lengths. An iRNA molecule can have one or more blunt ends, in which the ends of the non-projecting duplex region, and the strands are base-paired at the end of the duplex region.

An iRNA molecule of this disclosure may have one or more blunt ends, or it may have one or more projections, or it may have a combination of a blunt end and a projection end.

A 5 'end of a strand of an iRNA molecule may be at a blunt end, or it may be on a projection. A 3 'end of a strand of an iRNA molecule may be at a blunt end, or it may be on a projection.

A 5 'end of a strand of an iRNA molecule can be at a blunt end, while the 3' end is at a projection. A 3 'end of a strand of an iRNA molecule can be at a blunt end, while the 5' end is at a projection.

In some aspects, both ends of an iRNA molecule are blunt ends.

In additional aspects, both ends of an iRNA molecule have a projection.

The projections at the ends 5 'and 3 can be of different lengths.

In certain aspects, an iRNA molecule may have a blunt end in which the 5 'end of the antisense strand and the 3' end of the sense strand do not have any projecting nucleotides.

In additional aspects, an iRNA molecule may have a blunt end in which the 3 'end of the antisense strand and the 5' end of the sense strand do not have any projecting nucleotides.

An iRNA molecule can have base-pairing mismatches in the duplex region. Any nucleotide in a projection of an iRNA molecule can be a deoxyribonucleotide, or a ribonucleotide.

One or more deoxyribonucleotides may be at the 5 'end, where the 3' end of the other strand of the iRNA molecule may not have a projection, or may not have a deoxyribonucleotide projection.

One or more deoxyribonucleotides may be at the 3 'end, where the 5' end of the other strand of the iRNA molecule may not have a projection, or may not have a deoxyribonucleotide projection.

In some aspects, one or more, or all of the projection nucleotides of an iRNA molecule can be 2'-deoxyribonucleotides.

Dicer substrate iRNA molecules

In some aspects, an iRNA molecule can be of suitable length as a Dicer substrate, which can be processed to produce a RISC-active iRNA molecule. See, for example, Rossi et al., US2005 / 0244858.

A double-stranded RNA (dsRNA) that is a substrate for Dicer may be of sufficient length to be processed by Dicer to produce an active iRNA molecule, and may further include one or more of the following properties: (i) the dsRNA of the Dicer substrate may be asymmetric, for example, having a 3 'overhang on the antisense strand, and (ii) the dicer substrate dsRNA may have a modified 3' end on the sense strand to direct the orientation of the binding of Dicer and the processing of dsRNA to an active iRNA molecule.

Methods of using iRNA molecules

The nucleic acid molecules and iRNA molecules comprised in the compositions of this invention, as defined in the claims, can be administered to a cell or tissue by direct application of the molecules, or with the molecules in combination with a carrier or diluent.

The nucleic acid molecules and iRNA molecules comprised in the compositions of this invention, as defined in the claims, can be delivered or administered to a cell, tissue, organ or subject by direct application of the molecules with a carrier or diluent, or any other delivery vehicle that acts to aid, promote, or facilitate entry into a cell, eg, viral sequences, viral material, or lipid or liposome formulations.

Nucleic acid molecules and iRNA molecules comprised in the compositions of this invention, as defined in the claims, can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be administered locally to relevant tissues ex vivo or in vivo by direct dermal application, transdermal application, or injection.

Delivery systems can include, for example, aqueous and non-aqueous gels, creams, emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, sprays, hydrocarbon bases, and powders, and may contain excipients such as solubilizers and enhancers. permeation.

An inhibitory nucleic acid composition or molecule of this disclosure can be administered within a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide formulations or compositions suitable for administering the compounds to patients suffering from a disease that is caused by excessive cell proliferation. Administration can begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, suppository. or oral administration. For example, therapeutic formulations can be in the form of liquid solutions or suspensions; for oral administration, the formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops or aerosols.

The compositions and methods of this disclosure may include an expression vector that includes a nucleic acid sequence that encodes at least one iRNA molecule of this invention in a manner that allows expression of the nucleic acid molecule.

The nucleic acid molecules and iRNA molecules of this disclosure can be expressed from transcription units inserted into DNA or RNA vectors. Recombinant vectors can be DNA plasmids or viral vectors. Viral vectors that provide for transient expression of nucleic acid molecules can be used.

For example, the vector may contain sequences that code for both strands of an iRNA molecule of a duplex, or for a single nucleic acid molecule that is self-complementary and thus forms an iRNA molecule. An expression vector can include a nucleic acid sequence that encodes two or more nucleic acid molecules.

A nucleic acid molecule can be expressed within cells from eukaryotic promoters. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA / RNA vector.

In some aspects, a viral construct can be used to introduce an expression construct into a cell, for transcription of a dsRNA construct encoded by the expression construct, wherein the dsRNA is active in RNA interference.

The lipid formulations can be administered to animals by intravenous, intramuscular or intraperitoneal injection, or orally or by inhalation or other methods known in the art.

Pharmaceutically acceptable formulations are known and can be used to deliver oligonucleotides.

In one aspect of the above method, the inhibitory nucleic acid molecule is administered at a dosage of about 5 to 500 mg / m2 / day, eg, 5, 25, 50, 100, 125, 150, 175, 200, 225, 250, 275 or 300 mg / m2 / day.

In some aspects, the inhibitory nucleic acid molecules of this disclosure are administered systemically in dosages of from about 1 to 100 mg / kg, eg, 1, 5, 10, 20, 25, 50, 75, or 100 mg / kg. kg.

In additional aspects, the dosage can range from about 25 to 500 mg / m2 / day.

Methods known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" Ed. AR Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.

Formulations for parenteral administration may contain, for example, excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin or hydrogenated naphthalenes. Biocompatible and biodegradable lactide polymer, lactide / glycolide copolymer or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems for inhibitory nucleic acid molecules include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example lactose, or they may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or they may be oily solutions for administration as nasal drops or as a gel.

The formulations can be administered to human patients in therapeutically effective amounts (eg, amounts that prevent, eliminate, or reduce a disease state) to provide therapy for a neoplastic disease or condition. The preferred dosage of a nucleotide oligomer of the invention may depend on variables such as the type and extent of the disorder, the general health of the particular patient, the formulation of the compound's excipients, and its route of administration.

All of the above methods for reducing malignant tumors can be either an in vitro method or an in vivo method. The dosage can be determined by an in vitro test using cultured cells, etc., as is known in the art. An effective amount can be an amount that reduces tumor size in KRAS-associated tumors by at least 10%, at least 20%, or at least 30%, or at least 40%, or at least as much. 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, up to 100% of the tumor size.

A pharmaceutical composition of this disclosure may be effective in treating a KRAS-associated disease. Examples of the diseases include a disease due to abnormal cell proliferation, a disease due to KRAS mutation, and a disease due to GST-rc overexpression.

Examples of the disease due to abnormal cell proliferation include malignant tumors, hyperplasia, keloid, Cushing's syndrome, primary aldosteronism, erythroplasia, polycythemia vera, leukoplakia, hyperplastic scar, lichen planus, and lentiginosis.

Examples of the disease due to the KRAS mutation include malignant tumor (also called cancer or malignancy).

Examples of the disease due to GST-rc overexpression include malignant tumor.

Examples of cancer include sarcomas such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma and osteosarcoma, carcinomas such as brain tumor, head and neck carcinoma, carcinoma of the head and neck breast, lung carcinoma, carcinoma of the esophagus, carcinoma of the stomach, carcinoma of the duodenum, carcinoma of the appendix, carcinoma of the colon, carcinoma of the rectum, carcinoma of the liver, carcinoma of the kidney, carcinoma of the pancreas, carcinoma of the gallbladder, carcinoma of the ducts biliary, renal carcinoma, ureter carcinoma, bladder carcinoma, prostate carcinoma, testicular carcinoma, uterus carcinoma, ovarian carcinoma, skin carcinoma, leukemia and malignant lymphoma.

Cancer includes epithelial malignancy and non-epithelial malignancy. Cancer can be present anywhere in the body

In one aspect of the present disclosure, cancer includes cancer cells that have the mutated KRAS defined above. In another aspect, cancer includes cancer cells that exhibit proliferation independent of hormones or growth factors. In additional aspects, a cancer includes cancer cells exhibiting GST-rc overexpression.

Additional active agents or drugs to suppress rc-GST

Examples of additional active agents to inhibit the activity of GST-pi include, but are not limited to, substances that bind to GST-pi, for example glutathione, glutathione analogs (for example, those described in WO95 / 08563, WO96 / 40205, WO99 / 54346), ketoprofen, indomethacin (see, for example, Hall et al., Cancer Res. 1989; 49 (22): 6265-8), ethacrynic acid, pyriprost (see for example, Tew et al., Cancer Res. 1988; 48 (13): 3622-5), anti-GST-pi antibodies and GST-rc dominant negative mutants. These agents are either commercially available or can be appropriately produced on the basis of publicly known techniques.

Three-component formulations for agent delivery in a malignant tumor

As used herein, a component of a formulation, such as a "lipid", can be a single compound or it can be a combination of one or more suitable lipid compounds. For example, "a stabilizing lipid" can refer to a single stabilizing lipid or a combination of one or more suitable stabilizing lipids. One skilled in the art can readily appreciate that certain combinations of the compounds described herein can be used without undue experimentation, and that the description of one component of a formulation encompasses various combinations of compounds.

This disclosure can provide a composition for use in delivering an active agent to cells, tissues or organs, organisms and subjects, wherein the composition includes one or more ionizable lipid molecules of this invention.

The compositions of this invention can include one or more of the ionizable lipid molecules, together with a structural lipid and one or more lipids to reduce the immunogenicity of the nanoparticles.

An ionizable lipid molecule of this invention can be any mole% of a composition of this invention.

The ionizable lipid molecules of a composition of this disclosure can be from 50% by mole to 80% by mole of the lipid components of the composition. In certain aspects, the ionizable lipid molecules of a composition can be from 55 mol% to 65 mol% of the lipid components of the composition. In additional aspects, the ionizable lipid molecules of a composition can be about 60% by moles of the lipid components of the composition.

The structural lipid of a composition of this disclosure can be from 20% by mole to 50% by mole of the lipid components of the composition. In certain aspects, the structural lipid of a composition can be from 35% by mole to 45% by mole of the lipid components of the composition.

The one or more lipids to reduce the immunogenicity of the nanoparticles can be from a total of 1% by mole to 8% by mole of the lipid components of the composition. In certain aspects, the one or more lipids to reduce the immunogenicity of the nanoparticles can be from a total of 1% by mole to 5% by mole of the lipid components of the composition.

In additional aspects, a composition of this disclosure can further include a cationic lipid, which can be from 5% by mole to 25% by mole of the lipid components of the composition. In certain aspects, a composition of this invention can further include a cationic lipid, which can be from 5 mol% to 15 mol% of the lipid components of the composition. In these aspects, the molar ratio of cationic lipid concentrations to ionizable lipid molecules of a composition of this disclosure can be from 5:80 to 25:50.

In compositions of this invention, all of the lipid components may include one or more of the ionizable lipid molecular components, one or more structural lipids, and one or more lipids to reduce the immunogenicity of the nanoparticles.

Formulations with four components for the administration of agents in a malignant tumor

This disclosure can provide a composition for use in delivering an active agent to cells, tissues or organs, organisms and subjects, wherein the composition includes one or more ionizable lipid molecules of this disclosure.

The compositions of this invention can include one or more of the ionizable lipid molecules, together with a structural lipid, one or more stabilizing lipids, and one or more lipids to reduce the immunogenicity of the nanoparticles.

An ionizable lipid molecule of this invention can be any mole% of a composition of this invention.

The ionizable lipid molecules of a composition of this disclosure can be from 15 mol% to 40 mol% of the lipid components of the composition. In certain aspects, the ionizable lipid molecules of a composition can be from 20% by mole to 35% by mole of the lipid components of the composition. In additional aspects, the ionizable lipid molecules of a composition can be from 25% by mole to 30% by mole of the lipid components of the composition.

The structural lipid of a composition of this disclosure can be from 25% by mole to 40% by mole of the lipid components of the composition. In certain aspects, the structural lipid of a composition can be from 30% by mole to 35% by mole of the lipid components of the composition.

The sum of the stabilizing lipids in a composition of this disclosure can be from 25% by mole to 40% by mole of the lipid components of the composition. In certain aspects, the sum of the stabilizing lipids in a composition can be from 30% by mole to 40% by mole of the lipid components of the composition.

In some aspects, a composition of this invention may include two or more stabilizing lipids, wherein each of the stabilizing lipids individually may be from 5 mol% to 35 mol% of the lipid components of the composition. . In certain aspects, a composition of this disclosure may include two or more stabilizing lipids, wherein each of the stabilizing lipids individually may be from 10 mol% to 30 mol% of the lipid components of the composition. .

In certain aspects, the sum of the one or more stabilizing lipids can be from 25% by moles to 40% by moles of the lipids of the composition, in which each of the stabilizing lipids individually can be from 5 % by mole to 35% by mole.

In certain aspects, the sum of the one or more stabilizing lipids can be from 30% by moles to 40% by moles of the lipids of the composition, in which each of the stabilizing lipids individually can be from 10 % by moles up to 30% by moles.

The one or more lipids for reducing the immunogenicity of the nanoparticles can be from a total of 1% by mole to 8% by mole of the lipid components of the composition. In certain aspects, the one or more lipids to reduce the immunogenicity of the nanoparticles can be from a total of 1% by mole to 5% by mole of the lipid components of the composition.

In additional aspects, a composition of this disclosure can further include a cationic lipid, which can be from 5% by mole to 25% by mole of the lipid components of the composition. In certain aspects, a composition of this invention can further include a cationic lipid, which can be from 5 mol% to 15 mol% of the lipid components of the composition. In these aspects, the molar ratio of cationic lipid concentrations to ionizable lipid molecules of a composition of this disclosure can be from 5:35 to 25:15.

In compositions of this invention, all of the lipid components may include one or more of the ionizable lipid molecular components, one or more structural lipids, one or more stabilizing lipids, and one or more lipids to reduce the immunogenicity of the nanoparticles.

Examples of lipid compositions

In some embodiments, three lipid-like components, ie, one or more ionizable molecules, a structural lipid, and one or more lipids to reduce the immunogenicity of the nanoparticles can be 100% of the lipid components of the composition. In certain aspects, a cationic lipid can be included.

Examples of compositions of this invention, as defined in the claims, are shown in Table 9.

Table 9: compositions of lipid components (each in mole% of the total)

In certain aspects, four lipid-like components, that is, one or more ionizable lipid molecules, a structural lipid, one or more stabilizing lipids, and one or more lipids to reduce the immunogenicity of nanoparticles can be 100% of the components. lipids of the composition.

Examples of compositions of this invention, as defined in the claims, are shown in the table

Table 10: Compositions of lipid components (each in mole% of the total)

Ionizable lipid-like molecules

Examples of an ionizable lipid include compounds having the structure shown in formula I

R1 = CH2 (CH2) nOC (= O) R4

R2 = CH2 (CH2) mOC (= O) R5

where n and m are from 1 to 2; and R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group having from zero to two double bonds;

wherein R3 is selected from

in which

R6 is selected from H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

R7 is selected from H, alkyl, hydroxyalkyl;

Q is O NR7;

p is from 1 to 4.

Examples of an ionizable lipid include the following compound:

which is (9Z, 9'Z, 12Z, 12'Z) -bis (octadeca-9,12-dienoate) of ((2 - ((3S, 4R) -3,4-dihydroxypyrrolidin-1-yl) acetyl) azanodiyl) bis (ethane-2,1-diyl).

Examples of an ionizable lipid include the following compound:

which is ((2- (3- (hydroxymethyl) azetidin-1-yl) acetyl) azanodiyl) bis (ethane-2,1-diyl) ditetradecanoate.

Examples of an ionizable lipid include the following compound:

which is (9Z, 9'Z, 12Z, 12'Z) -bis (octadeca-9,12-dienoate) of ((2- (4- (2-hydroxyethyl) piperazin-1-yl) acetyl) azanodiyl) bis (ethane-2,1-diyl).

Examples of an ionizable lipid include the following compound:

which is (9Z, 9'Z, 12Z, 12'Z) -bis (octadeca-9,12-dienoate) of ((2- (4- (2-hydroxyethyl) piperazin-1-yl) acetyl) azanodiyl) bis (ethane-2,1-diyl).

Examples of an ionizable lipid include compounds having the structure shown in formula IV

R1 = C (= O) OR4

R2 = C (= O) OR5

wherein R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group having from zero to two double bonds;

wherein R3 is selected from

in which

R6 is selected from H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

R7 is selected from H, alkyl, hydroxyalkyl;

Q is O NR7;

p is from 1 to 4.

Examples of an ionizable lipid include the following compound:

which is 2 - ((1 - (((9Z, 12Z) -heptadeca-9,12-dien-1-yl) oxy) -5 - (((9Z, 12Z) -octadeca-9,12-dien-1 -yl) oxy) -1,5-dioxopentan-3-¡l) amino) -N, N, N-trimethyl-2-oxoethane-1-amime.

Examples of an ionizable lipid include compounds having the structure shown in formula VI

where R1 is

R1 = OC (= O) R4

wherein R2 and R4 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group having from zero to two double bonds;

wherein R3 is selected from aminoalkyl, quaternary aminoalkyl.

Examples of an ionizable lipid include the following compound:

which is (9Z, 12Z) -octadeca-9,12-dienoate of 2 - ((9Z, 12Z) -N- (3- (dimethylamine) prop¡l) octadeca-9,12-d Enam¡do) et¡lo. Examples of an ionizable lipid include the following compound:

which is N, N, N-tr¡met¡l-3 - ((9Z, 12Z) -N- (2 - (((9Z, 12Z) -octadeca-9,12-d¡eno¡l) ox¡ ) ethyl) octadeca-9,12-dieneamide) propan-1-aminium.

Examples of an ionizable lipid include compounds that have the structure shown in Formula IX.

where R1 and R2 are

R1 = C (= O) OR4

R2 = NHC (= O) R5

in which R4 and R5 are independently for each appearance a C (12-20) alkyl group or a C (12-20) alkenyl group having from zero to two double bonds;

where r is from 1 to 4;

in which R3 is selected from

in which

R6 is selected from H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

R7 is selected from H, alkyl, hydroxyalkyl;

Q is O NR7.

Examples of an ionizable lipid include the following compound:

which is N, N, N-trimethyl-2 - (((S) -3 - (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) -2 - ((9Z, 12Z ) -octadeca-9,12-dienamido) -3-oxopropyl) amino) -2-oxoethane-1-aminium.

Examples of an ionizable lipid molecule include compounds having the structure shown in formula III.

where R1 and R2 are

R1 = CH2 (CH2) nOC (= O) R4

R2 = CH2 (CH2) mOC (= O) R5

where n and m are from 1 to 2; and R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group;

wherein R3 is selected from alkyl, hydroxyalkyl, alkoxyalkoxy, and carboxyalkyl;

wherein R6 is selected from NR72, N + HR72, and N + R73;

wherein R7 is selected from H, alkyl, hydroxyalkyl.

Examples of an ionizable lipid molecule include compounds having the structure shown in formula IV-B

Fórmula IV-B

Formula IV-B

where R1 and R2 are

R1 = C (= O) OR4

R2 = C (= O) OR5

wherein R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group;

where Z is S or O;

wherein R3 is selected from

in which

each R6 is independently selected from H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

R7 is selected from H, alkyl, hydroxyalkyl;

p is from 1 to 4.

Examples of an ionizable lipid molecule include compounds having the structure shown in formula V

where R1 and R2 are

R1 = NHC (= O) R4

R2 = C (= O) OR5

wherein R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group;

where p is from 1 to 4;

wherein R3 is selected from

in which

each R6 is independently selected from H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

R7 is selected from H, alkyl, hydroxyalkyl;

Q is O NR7.

Examples of an ionizable lipid molecule include compounds having the structure shown in formula VII.

where R1 and R2 are

R1 = CH2 (CH2) nOC (= O) R4

R2 = CH2 (CH2) mOC (= O) R5

where n and m are from 1 to 2; and R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group;

wherein R3 is selected from aminoalkyl, quaternary aminoalkyl, alkoxyalkyl, alkoxyalkoxyalkyl.

Examples of an ionizable lipid molecule include compounds having the structure shown in formula VIII.

Fórmula VIII

Formula VIII

where R1 and R2 are

R1 = OC (= O) R4

R2 = C (= O) ZR5

where Z is NH or O,

wherein R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group;

wherein R3 is selected from

Not me;

quaternary amino;

aminoalkyl;

quaternary aminoalkyl;

NHC (= O) (CH2) pR8;

NHC (= O) SR9;

where R8 is selected from

carboxyalkyl;

aminoalkyl;

aminoalkyl;

and in which

each R6 is independently selected from H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

R7 is selected from H, alkyl, hydroxyalkyl;

Q is O NR7.

Examples of an ionizable lipid molecule include compounds that have the structure shown in the formula VIII-B

Fórmula VIII-B

Formula VIII-B

where R1 and R2 are

R1 = CH2 (CH2) nOC (= O) R4

R2 = CH2 (CH2) mOC (= O) R5

where n and m are from 1 to 2;

R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group; where Z is N, O;

wherein R3 is selected from

in which

each R6 is independently selected from H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

R7 is selected from H, alkyl, hydroxyalkyl;

p is from 1 to 4.

Examples of an ionizable lipid molecule include compounds having the structure shown in formula X

where R1 and R2 are

R1 = C (= O) OR4

R2 = NHC (= O) R5

wherein R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group;

wherein R3 is selected from

Not me;

quaternary amino;

aminoalkyl;

quaternary aminoalkyl;

hydroxyalkylamino;

quaternary hydroxyalkylamino.

Examples of an ionizable lipid molecule include compounds having the structure shown in formula XI

where R1 and R2 are

R1 = C (= O) R4

R2 = C (= O) OR5

wherein R4 and R5 are independently for each occurrence a C (12-20) alkyl group or a C (12-20) alkenyl group;

where p is from 1 to 4;

wherein R3 is selected from

in which

each R6 is independently selected from H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkoxy, aminoalkyl;

R7 is selected from H, alkyl;

Q is O NR7.

Structural lipids

Examples of structural lipids include cholesterols, sterols, and steroids.

Examples of structural lipids include colanes, cholestanes, ergostanes, campestanes, poriferastanes, stigmastanes, gorgostanos, lanostanos, gonanes, estranos, androstanos, preganes and cycloartanos.

Examples of structural lipids include sterols and zoosterols such as cholesterol, lanosterol, zimosterol, zimostenol, desmosterol, stigmastanol, dihydrolanosterol, and 7-dehydrocholesterol.

Examples of structural lipids include pegylated cholesterols and cholestane-3-oxo-acyl (C1-22) compounds, for example, cholesteryl acetate, cholesteryl arachidonate, cholesteryl butyrate, cholesteryl hexanoate, cholesteryl myristate, cholesteryl palmitate , cholesteryl behenate, cholesteryl stearate, cholesteryl caprylate, cholesteryl n-decanoate, cholesteryl dodecanoate, cholesteryl nervonate, cholesteryl pelargonate, cholesteryl n-valerate, cholesteryl oleate, cholesteryl elaidate, cholesteryl etheric acid of cholesteryl, cholesteryl linolelaidate and cholesteryl linoleate.

Examples of structural lipids include sterols such as phytosterols, beta-sitosterol, campesterol, ergosterol, brassicasterol, delta-7-stigmasterol, and delta-7-avenasterol.

Stabilizing lipids

Examples of stabilizing lipids include zwitterionic lipids.

Examples of stabilizing lipids include compounds such as phospholipids.

Examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylphostenylphosphate, diocholylphosphatidylphostenylphosphate

Examples of stabilizing lipids include phosphatidylethanolamine compounds and phosphatidylcholine compounds. Examples of stabilizing lipids include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

Examples of stabilizing lipids include diphytanoylphosphatidylethanolamine (DPhPE) and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC).

Examples of stabilizing lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sglycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

Examples of stabilizing lipids include 1,2-dilauroyl-sn-glycerol (DLG); 1,2-dimyristoyl-sn-glycerol (DMG); 1,2-dipalmitoyl-sn-glycerol (DPG); 1,2-distearoyl-sn-glycerol (DSG); 1,2-diaraquidoyl-sn-glycero-3-phosphocholine (Da PC); 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-dipalmitoyl-sn-glycero-O-ethyl-3-phosphocholine (DPePC); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DI PE); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (P-Smooth-PC); and 1-stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-Smooth-PC).

Lipids to reduce immunogenicity

Examples of lipids to reduce immunogenicity include polymeric compounds and conjugates. lipid polymer.

Examples of lipids to reduce immunogenicity include pegylated lipids having polyethylene glycol (PEG) regions. The PEG regions can be of any molecular mass. In some embodiments, a PEG region can have a molecular mass of 200, 300, 350, 400, 500, 550, 750, 1000, 1500, 2000, 3000, 3500, 4000, or 5000 Da.

Examples of lipids for reducing immunogenicity include compounds having a methoxypolyethylene glycol region.

Examples of lipids for reducing immunogenicity include compounds having a carbonylmethoxypolyethylene glycol region.

Examples of lipids for reducing immunogenicity include compounds having a multi-branched PEG region.

Examples of lipids for reducing immunogenicity include compounds that have a polyglycerin region. Examples of lipids to reduce immunogenicity include polymeric lipids such as DSPE-mPEG, DMPE-mPEG, DPPE-mPEG, and DOPE-mPEG.

Examples of lipids to reduce immunogenicity include PEG-phospholipids and PEG-ceramides.

Cationic lipids

Examples of cationic lipids include cationic HEDC compounds as described in US 2013/0330401 A1. Some examples of cationic lipids are provided in US 2013/0115274 A1. Additional examples of cationic lipids are known in the art.

Lipid compositions

In some aspects, a composition may contain the ionizable lipid compound 81, the structural lipid cholesterol, the stabilizing lipids DOPc and DOPe, and the lipid to reduce DPPE-mPEG immunogenicity. In certain aspects, Compound 81 can be 15 to 25% by mole of the composition; cholesterol, DOPC and DOPE combined can be 75 to 85% by mole of the composition; and DPPE-mPEG can be 5 mol% of the composition.

In one aspect, compound 81 can be 25% by mole of the composition; cholesterol can be 30 mol% of the composition, DOPC can be 20 mol% of the composition, DOPE can be 20 mol% of the composition; and DPPE-mPEG (2000) can be 5 mol% of the composition.

Nanoparticles

Aspects of this disclosure can provide liposome nanoparticle compositions. The ionizable molecules of this disclosure can be used to form liposome compositions, which can have a bilayer of lipid-like molecules.

A nanoparticle composition can have one or more of the ionizable molecules of this invention in a liposomal structure, a bilayer structure, a micelle, a lamellar structure, or a mixture thereof. In some aspects, a composition can include one or more liquid carrier components. A suitable liquid vehicle for the administration of active agents of this invention can be a pharmaceutically acceptable liquid vehicle. A liquid carrier can include an organic solvent or a combination of water and an organic solvent.

Aspects of this disclosure can provide lipid nanoparticles that are from 10 to 1000 nm in size. In some aspects, the liposomal nanoparticles can be from 10 to 150 nm in size.

In certain aspects, the liposomal nanoparticles of this disclosure can encapsulate the iRNA molecule and retain at least 80% of the encapsulated iRNA molecules after 1 hour of exposure to human serum.

Pharmaceutical compositions

This disclosure further contemplates methods of delivering an active agent to an organ of a subject to treat a malignant tumor by administering to the subject a composition of this invention. Organs that can be treated include the lung, liver, pancreas, kidney, colon, bone, skin, and intestine.

In some aspects, this disclosure provides methods of treating a malignant lung tumor disease by administering a composition of this invention to the subject.

In additional aspects, this disclosure provides a range of pharmaceutical formulations.

A pharmaceutical formulation herein may include an active agent, as well as a drug carrier, or a lipid of this invention, together with a pharmaceutically acceptable carrier or diluent. In general, the active agents of this disclosure include any malignant tumor active agent, including any inhibitory nucleic acid molecule and any small molecular drug. Examples of inhibitory nucleic acid molecules include ribozymes, antisense nucleic acids, and RNA interference molecules (iRNA molecules).

Examples of antitumor and antineoplastic drugs include oncogene-related factors, tumor angiogenesis factor, metastasis-related factors, cytokines such as TGF-beta, growth factor, proteinase such as MMP, caspase, and immunization suppression factor.

A drug carrier can direct a composition to reach stellate cells. A drug carrier may include a drug within, or be attached to the exterior of a drug-containing substance, or mixed with a drug as long as a retinoid derivative and / or a vitamin A analog is included in the carrier of drugs, and is at least partially exposed on the outside of the preparation. The composition or preparation can be covered with an appropriate material, such as, for example, an enteric coating or a material that disintegrates over time, or it can be incorporated into an appropriate drug delivery system.

A pharmaceutical formulation of this disclosure may contain one or more of each of the following: a surfactant, a diluent, an excipient, a preservative, a stabilizer, a dye, and a suspending agent.

Certain pharmaceutical carriers, diluents, and components for a pharmaceutical formulation, as well as methods for formulating and administering the compounds and compositions of this invention are described in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Penn. (1990).

Examples of preservatives include sodium benzoate, ascorbic acid, and p-hydroxybenzoic acid esters.

Examples of surfactants include alcohols, esters, sulfated aliphatic alcohols.

Examples of excipients include sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium aluminate-metasilicate, synthetic aluminum silicate, calcium carbonate, sodium bicarbonate, calcium hydrogen phosphate, and carboxymethyl cellulose. calcium.

Examples of suspending agents include coconut oil, olive oil, sesame oil, peanut oil, soybean, cellulose acetate-phthalate, methyl acetate-methacrylate copolymer, and ester phthalates.

A therapeutic formulation of this disclosure for the administration of one or more active molecules for gene silencing can be administered to a mammal in need. A therapeutically effective amount of the formulation and active agent, which can be encapsulated in a liposome, can be administered to a mammal to prevent or treat a malignant tumor.

The route of administration can be local or systemic.

A therapeutically effective formulation of this invention can be administered by a variety of routes, including intravenous, intraperitoneal, intramuscular, subcutaneous, and oral.

Routes of administration may include, for example, parenteral administration, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.

The formulation can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, and the like, for prolonged and / or timed repeat administration at a predetermined rate.

The composition of the present disclosure can be administered via various routes, including oral and parenteral routes, and examples thereof include, but are not limited to, oral, intravenous, intramuscular, subcutaneous, local, intrapulmonary, intrarespiratory, intratracheal, intrabronchial, nasal, rectal, intraarterial, intraportal, intraventricular, intramedullary, intraganglionic, intralymphatic, intracerebral, intrathecal, intracerebroventricular, transmucosal, percutaneous, intranasal, intraperitoneal and intrauterine, and can be formulated in a dosage form suitable for each route of administration. Such a dosage form and formulation method can be selected as appropriate from any known dosage form and method. See, for example, Hyojun Yakuzaigaku, Standard Pharmaceutics, ed. by Yoshiteru Watanabe et al., Nankodo, 2003.

Examples of dosage forms suitable for oral administration include, but are not limited to, powder, granule, tablet, capsule, liquid, suspension, emulsion, gel, and syrup, and examples of dosage forms suitable for parenteral administration include injections such as an injectable solution, an injectable suspension, an injectable emulsion, and a ready-to-use injection. Formulations for parenteral administration may be in a form such as a sterile isotonic aqueous or non-aqueous solution or suspension.

Pharmaceutical formulations for parenteral administration, eg, by bolus injection or continuous infusion, include aqueous solutions of the active formulation in water-soluble form. Suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions.

Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The formulations may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain agents from a formulary such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg sterile pyrogen-free water, before use.

In addition to the preparations described above, the formulations can also be formulated as a depot preparation. Such long-acting formulations can be administered by intramuscular injection. Thus, for example, the formulation can be formulated with suitable polymeric or hydrophobic materials, for example, as an emulsion in an acceptable oil, or ion exchange resins, or as poorly soluble derivatives, for example, as a poorly soluble salt.

The compositions of this invention and the formulations disclosed herein can also be formulated for topical administration and can be applied to the skin of the subject using any method suitable for application of the vehicle of topical administration. For example, the formulation can be applied manually, using an applicator, or by a procedure that involves both. After application, the formulation can be made to act on the skin of the subject, eg, by rubbing. The application can be done several times a day or once a day. For example, the formulation can be applied to a subject's skin once a day, twice a day, or several times a day, or it can be applied once every two days, once every three days, or about once a week, once every two weeks or once every several weeks.

The pharmaceutical formulations or compositions described herein can be administered to the subject by any suitable means. Examples of methods of administration include, but are not limited to, (a) administration via injection, subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, intraorbital, intracapsular, intraspinal, intrasternal or the like, including infusion pump administration; (b) administration locally such as by injection directly into the renal or cardiac area, for example, by depot implantation; as well as those skilled in the art deem it appropriate to contact the active compound with living tissue.

The individual physician can choose the exact formulation, route of administration and dosage for the pharmaceutical compositions in light of the patient's condition. See, for example, Goodman & Gilman, The Pharmacological Basis of Therapeutics, 12th ed., Sec. 1, 2011. Typically, the dosage range of the composition administered to the patient may be from about 0.5 to about 1000 mg / kg of the patient's body weight. Dosage may be a single or a series of two or more administered over the course of one or more days, as needed by the patient. In cases where human dosages for compounds have been established for at least some state, the dosages will be about the same, or dosages that will be about 0.1% to about 500%, more preferably about 25%. at approximately 250% of the established human dosage. When no human dosage is established, as will be the case with newly discovered pharmaceutical compositions, a suitable human dosage can be inferred from ED50 or ID50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified. toxicity studies and efficacy studies in animals.

Methods to prevent or treat a malignant tumor

The present disclosure further relates to a method for monitoring the activity or growth of tumors. malignancies, the method including administering an effective amount of the composition to a subject in need thereof. The effective amount referred to herein, in a method of treating a malignant tumor, alleviates its symptoms or slows or arrests its progression, and is preferably an amount that prevents the appearance or recurrence of a malignant tumor, or so cure. It is also preferably an amount that does not cause any adverse effects that exceed the benefit of administration. Such an amount can be determined as appropriate by an in vitro test using cultured cells or by a test on a model animal or mammal such as a mouse, rat, dog or pig, and such test methods are well known to one of skill in the art. Furthermore, the dose of the active agents in the carrier and the dose of the active agents used in the method of the present disclosure are known to one skilled in the art, or can be determined as appropriate by the tests mentioned above.

The frequency of administration depends on the properties of the composition used and the conditions of the subject mentioned above, and can be a plurality of times a day (i.e. 2, 3, 4, 5 or more times a day), once a day, every few days (i.e. every 2, 3, 4, 5, 6 or 7 days, etc.), a few times a week (for example, 2, 3, 4 times, etc. a week) , every two weeks or every few weeks (i.e. every 2, 3, 4 weeks, etc.).

In some aspects, the present disclosure also relates to a method of delivering a drug to a malignant tumor cell, using the above carrier. This method includes a step of administering or adding the carrier having the substance to be administered to a living being or to a medium, for example, a culture medium, containing a cell that produces extracellular matrix in the lung. These steps can be accomplished as appropriate according to any known method or a method described in this invention. Furthermore, the above method includes a mode carried out in vitro and a mode in which a malignant tumor cell in the lung is targeted within the body.

A therapeutically effective formulation of this disclosure can be administered by systemic administration that can provide broad biodistribution of the active agent.

Aspects of this disclosure can provide a therapeutic formulation, which includes a therapeutic molecule of the invention and a pharmaceutically acceptable carrier.

An effective dose of a formulation of this invention can be administered from 1 to 12 times a day or once a week. The duration of administration can be 1, 2, 3, 4, 5, 6 or 7 days, or it can be 1,2, 3, 4, 5, 6, 8, 10 or 12 weeks.

Additional aspects

A composition for use in delivering an active agent for treating a malignant tumor in a subject, the composition comprising an ionizable lipid, a structural lipid, and a lipid to reduce the immunogenicity of the nanoparticles. The malignant tumor can be located in the lung, colon, kidney, or pancreas. The malignant tumor can be located in the liver, bones, skin, or intestine. The ionizable lipid can be from 50% by mole to 80% by mole of the lipids in the composition, or from 55% by mole to 65% by mole of the lipids in the composition. The structural lipid can be from 20% by moles to 50% by moles of the lipids of the composition, or from 35% by moles to 55% by moles of the lipids of the composition. The structural lipid can be cholesterol, sterol, or steroid. The lipid for reducing the immunogenicity of the nanoparticles can be from 1% by mole to 8% by mole of the lipids of the composition. The lipid to reduce the immunogenicity of the nanoparticles may have a polyethylene glycol (PEG) region having a molecular mass of from 200 to 5000 Da. The lipid to reduce the immunogenicity of the nanoparticles can be DPPE-mPEG, DSPE-mPEG, DMPE-mPEG or DOPE-mPEG.

This disclosure further contemplates a composition for use in delivering an active agent for treating a malignant tumor in a subject, the composition comprising an ionizable lipid, a structural lipid, one or more stabilizing lipids, and a lipid for reducing immunogenicity. of nanoparticles. The ionizable lipid can be from 15 mol% to 40 mol% of the lipids of the composition, or from 20 mol% to 35 mol% of the composition lipids. The structural lipid can be from 25 mol% to 40 mol% of the lipids of the composition, or from 30 mol% to 35 mol% of the composition lipids.

The sum of the one or more stabilizing lipids can be from 25% by moles to 40% by moles of the lipids of the composition, in which each of the stabilizing lipids individually is from 5% by moles to the 35% in moles. The one or more stabilizing lipids can be phosphatidylethanolamine compounds or phosphatidylcholine compounds.

The composition can be (compound 81 / cholesterol / DOPC / DOPE / DPPE-PEG-2000) in one of the following combinations, in which the numbers refer to the concentration in mole% of the component: (25/30/30 / 10/5), (25/30/25/15/5), (25/30/20/20/5), (25/30/15/25/5), (25/30/10/30 / 5), (25/35/15/20/5), (25/35/20/15/5), (30/30/15/20/5), (30/30/20/15/5 ), (35/30/15/15/5). In some aspects, the ionizable lipid may be compound 81, the lipid The structural lipid can be cholesterol, the stabilizing lipids can be DOPC and DOPE and the lipid to reduce the immunogenicity of the nanoparticles can be DSPE-mPEG-2000, in which the compound 81 comprises 15 to 25% in moles of the composition, in wherein the cholesterol, DOPC, and DOPE combined comprise 75 to 85% by mole of the composition, wherein DSPE-mPEG-2000 comprises from 1 to 5% by mole of the composition, and wherein compound 81 Combined, cholesterol, DOPC, DOPE, and DSPE-mPEG-2000 comprise substantially 100 mole% of the lipids in the composition.

The active agent can be GST-rc targeting iRNA molecules and p21 targeting iRNA molecules. In some aspects, the active agent comprises GST-rc targeting iRNA molecules and p21 targeting iRNA molecules, and the composition comprises liposomal nanoparticles that encapsulate the iRNA molecules. In certain aspects, the active agent comprises GST-rc targeting iRNA molecules and p21 targeting iRNA molecules, and the compositions comprise liposomal nanoparticles that encapsulate the iRNA molecules and retain at least 80% of the subsequently encapsulated iRNA molecules. of 1 hour of exposure to human serum.

This disclosure also contemplates pharmaceutical compositions comprising a lipid composition and an active agent. The active agent can be one or more iRNA molecules. IRNA molecules for treating a malignant tumor can include GST-rc targeting iRNA molecules and p21 targeting iRNA molecules. The iRNA molecules for treating a malignant tumor can be, for example, siRNA, shRNA or micro-RNA, as well as RNA-directed RNA (ddRNA), Piwi-interacting RNA (piRNA) and repeat-associated siRNA (rasiRNA).

In certain aspects, this disclosure provides a pharmaceutical composition containing iRNA molecules for treating a malignant tumor which are iRNA molecules targeting only GST-rc.

Aspects of this disclosure further include methods of delivering an active agent to an organ of a subject to treat a malignancy, by administering a composition of this invention to the subject. The malignant tumor can be located in the lung, colon, kidney, or pancreas. The malignant tumor can be located in the liver, bones, skin, or intestine. The malignant tumor can be located in a selected anatomical region from the group consisting of head and neck, brain, breast, esophagus, stomach, intestine, duodenum, liver, gallbladder, bile ducts, kidney, urethra, bladder, prostate, testicle, uterus , ovary, skin, bone, bone marrow, blood, skin and epithelial layer.

Administration can be a dose of from 0.01 to 2 mg / kg of the iRNA molecules at least once a day for a period of up to twelve weeks. Administration can provide a mean (0-last) AUC of from 1 to 1000 ug * min / ml and a mean Cmax of from 0.1 to 50 ug / ml for the GST-rc iRNA molecule. Administration can provide a mean (0-last) plasma AUC of from 1 to 1000 ug * min / ml and a mean plasma Cmax of from 0.1 to 50 ug / ml for the p21 iRNA molecule.

This invention provides compositions, as defined in the claims, for use in methods of preventing, treating or ameliorating one or more symptoms of a malignant tumor in a mammal in need thereof, or any animal, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising iRNA molecules, wherein a portion of the iRNA molecules are active in reducing GST-rc expression and a portion of the iRNA molecules are active in reducing p21 expression. In some embodiments, the malignant tumor is associated with the KRAS mutation in the mammal, the method further comprising identifying a tumor cell in the mammal, the tumor cell comprising at least one of: (i) a KRAS gene mutation and (ii ) an aberrant expression level of the KRAS protein.

The methods of this disclosure can be applied to any animal, including humans.

The mammal can be a human, the GST-rc can be a human GST-rc, and the p21 can be a human p21. In some aspects, the malignant tumor overexpresses GST-rc, and if GST-rc expression is down-regulated, then the level of p21 can be up-regulated. IRNA molecules can decrease the expression of GST-rc and p21 in the mammal. Administration can decrease the expression of GST-rc and p21 in the mammal by at least 5% for at least 5 days. The method can reduce one or more symptoms of the malignant tumor, delay or interrupt the progression of the malignant tumor, or reduce the growth of malignant tumor cells in the subject. Tumor cells may comprise increased expression levels of the wild-type KRAS protein compared to that of a normal cell. Tumor cells may overexpress wild-type GST-rc protein or RNA, and if GST-rc expression is suppressed, the level of p21 protein or RNA may be elevated.

In some aspects, the tumor cells may comprise mutations in the KRAS protein at one or more of residues 12, 13, and 61. In certain aspects, the tumor cells may comprise mutations in the KRAS protein and the tumor is a cancer selected from cancer colon, pancreatic cancer and lung cancer. The tumor may be a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, and colorectal carcinoma. The malignant tumor can be a sarcoma selected from the group of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, colorectal carcinoma, breast cancer, and fibrosarcoma. The malignant tumor can be located in any anatomical region, which in some aspects can be selected from the group of lung, liver, pancreas, colon, kidney, bone, skin, intestine and any combination thereof.

In another aspect, this invention relates to the surprising discovery that the size of malignant tumors can be reduced in vivo by treatment with compositions comprising a pharmaceutically acceptable carrier and siRNA inhibitors of GST-rc and p21. Furthermore, this invention provides the unexpectedly advantageous result that tumor cell apoptosis can be increased to a level of synthetic lethality to provide methods and compositions for preventing or treating malignant tumors.

This invention relates to compositions and their use in methods incorporating nucleic acid-based therapeutics for use in administration to various organs to prevent, treat, or ameliorate malignant tumor conditions and diseases. In some aspects, this disclosure provides compositions of RNA interference molecules (iRNA molecules) for gene silencing of various malignant tumor-related targets.

This disclosure can provide compositions for delivering therapeutic molecules, as well as methods of using the same. Various pharmacological and RNA-based compositions of this disclosure can be used in methods for preventing or treating malignant tumors.

In some embodiments, malignant tumors that contain a KRAS mutation or that show aberrant KRAS expression levels can be reduced by treatment with compositions comprising a pharmaceutically acceptable carrier and siRNA agents that modulate GST-rc and p21 expression. a level that creates synthetic lethality for tumor cells.

This invention relates to compositions and their use in methods for nucleic acid-based therapeutics against malignant tumors. In some embodiments, this invention provides compositions comprising a pharmaceutically acceptable carrier and iRNA molecules, structures, and compositions that can silence GST-rc and p21 expression. The structures and compositions of this disclosure can be used in the prevention, treatment, or reduction of the size of malignant tumors.

This disclosure provides compositions and methods that can be used to treat a neoplasm in a subject. In particular, this disclosure provides therapeutic compositions that can decrease the expression of a GST-rc nucleic acid molecule or polypeptide, as well as a p21 nucleic acid molecule or polypeptide to treat a KRAS-associated neoplasia.

In some aspects, this disclosure includes an inhibitory nucleic acid molecule that corresponds to, or is complementary to, at least one fragment of a GST-rc nucleic acid molecule, and that decreases GST-rc expression in a cell. In additional aspects, this disclosure includes an inhibitory nucleic acid molecule that corresponds to, or is complementary to, at least one fragment of a p21 nucleic acid molecule and that decreases the expression of p21 in a cell.

In certain aspects, this disclosure provides double-stranded nucleic acid molecules that are iRNA molecules such as siRNA or shRNA, as well as DNA-directed RNA (ddRNA), Piwi-interacting RNA (piRNA), and repeat-associated siRNA (rasiRNA) to delete GST-rc and p21.

The methods of this disclosure can be applied to any animal, including humans.

In some aspects, this disclosure includes one or more vectors that encode the inhibitory nucleic acid molecules described above. A vector can be a retroviral, adenoviral, adeno-associated viral, or lentiviral vector. In additional aspects, a vector may contain a promoter suitable for expression in a mammalian cell. Additional aspects include cancer cells that contain a KRAS mutation or that show aberrant KRAS expression levels, which may also contain the vector, or an inhibitory nucleic acid molecule according to any one of the above aspects. In additional aspects, the cells can be neoplastic cells in vivo.

In some aspects, this disclosure includes methods for decreasing the expression of GST-rc and p21 in a malignant tumor cell that contains a KRAS mutation or exhibits aberrant KRAS expression. The methods may include contacting the cell with an effective amount of inhibitory nucleic acid molecules, wherein the inhibitory nucleic acid molecules inhibit the expression of a GST-rc polypeptide and a p21 polypeptide, thereby decreasing the expression of GST-rc and p21 in the cell.

In additional aspects, the methods of this disclosure can decrease the transcription or translation of GST-rc and p21 in malignant tumors.

In particular aspects, this disclosure includes methods for decreasing the expression of GST-rc and p21 in a malignant tumor cell, wherein the cell can be a human cell, a neoplastic cell, an in vivo cell, or a cell in vitro.

Aspects of this disclosure may also provide methods for treating a subject having a neoplasm, wherein the cancer cells of the neoplasm contain a KRAS mutation or show aberrant KRAS expression levels. The methods may involve administering to the subject an effective amount of two or more inhibitory nucleic acid molecules, wherein the inhibitory nucleic acid molecules reduce the expression of GST-rc and p21, thereby treating the neoplasia. In some aspects, the methods of this disclosure can reduce the size of a neoplasm, relative to the size of a neoplasm before treatment or without treatment.

In various aspects, an inhibitory nucleic acid molecule can be delivered in a liposome, a polymer, a microsphere, a nanoparticle, a gene therapy vector, or a naked DNA vector.

In further aspects, this disclosure presents methods for treating a subject, eg, a human patient, having a neoplasm in which the cancer cells in the neoplasm contain a KRAS mutation or show aberrant KRAS expression levels. In certain aspects, the methods may include administering to the subject an effective amount of inhibitory nucleic acid molecules, wherein the inhibitory nucleic acid molecules are antisense nucleic acid molecules, siRNA, dsRNA that are active for RNA interference, or a combination thereof, which inhibits the expression of a GST-rc polypeptide and a p21 polypeptide.

In particular aspects, a neoplasm cell overexpressed GST-rc and, if GST-rc is suppressed, the level of p21 may be elevated.

In certain aspects, the neoplasm can be a malignant tumor or lung cancer, kidney cancer, or pancreatic cancer.

Structures of lipid tails

A lipid-like compound of this disclosure may have one or more lipophilic tails containing one or more alkyl or alkenyl groups. Examples of lipophilic tails include C alkenyl (14: 1 (5)), C alkenyl (14: 1 (9)), C alkenyl (16: 1 (7)), C alkenyl (16: 1 (9)), C (18: 1 (3)) alkenyl, C (18: 1 (5)) alkenyl, C (18: 1 (7)) alkenyl, C (18: 1 (9)) alkenyl, C (18: 1) alkenyl (11)), C alkenyl (18: 1 (12)), C alkenyl (18: 2 (9.12)), C alkenyl (18: 2 (9.11)), C alkenyl (18: 3 (9 , 12.15)), C alkenyl (18: 3 (6,9,12)), C alkenyl (18: 3 (9,11,13)), C alkenyl (18: 4 (6,9,12, 15)), C alkenyl (18: 4 (9,11,13,15)), C alkenyl (20: 1 (9)), C alkenyl (20: 1 (11)), C alkenyl (20: 2 ( 8.11)), C alkenyl (20: 2 (5.8)), C alkenyl (20: 2 (11.14)), C alkenyl (20: 3 (5.8.11)), C alkenyl ( 20: 4 (5,8,11,14)), C alkenyl (20: 4 (7,10,13,16)), C alkenyl (20: 5 (5,8,11,14,17)), C alkenyl (20: 6 (4,7,10,13,16,19)), C alkenyl (22: 1 (9)), C alkenyl (22: 1 (13)) and C alkenyl (24: 1 ( 9)).

Chemical definitions

The term "alkyl", as used herein, refers to a hydrocarbyl radical of a saturated aliphatic group, which can be of any length. An alkyl group can be a branched or unbranched, substituted or unsubstituted aliphatic group containing from 1 to 22 carbon atoms. For example, this definition also applies to the alkyl portion of other groups such as, for example, cycloalkyl, alkoxy, alkanoyl, and aralkyl.

As used herein, a term such as "C (1-5) alkyl" includes C (1) alkyl, C (2) alkyl, C (3) alkyl, C (4) alkyl, and C (4) alkyl. 5). Similarly, for example, the term "C (3-22) alkyl" includes C (1) alkyl, C (2) alkyl, C (3) alkyl, C (4) alkyl, C (5) alkyl, alkyl C (6), C (7) alkyl, C (8) alkyl, C (9) alkyl, C (10) alkyl, C (11) alkyl, C (12) alkyl, C (13) alkyl, C ( 14), C (15) alkyl, C (16) alkyl, C (17) alkyl, C (18) alkyl, C (19) alkyl, C (20) alkyl, c (21) alkyl, and C (22) alkyl .

As used herein, an alkyl group can be designated by a term such as Me (methyl), Et (ethyl), Pr (any propyl group), nPr (n-Pr, n-propyl), iPr (i -Pr, isopropyl), Bu (any butyl group), nBu (n-Bu, n-butyl), Bu (i-Bu, isobutyl), sBu (s-Bu, sec-butyl) and 'Bu (t-Bu , tert-butyl).

The term "alkenyl", as used herein, refers to a hydrocarbyl radical having at least one carbon-carbon double bond. An alkenyl group can be a branched or unbranched, substituted or unsubstituted hydrocarbyl radical having 2 to 22 carbon atoms and at least one carbon-carbon double bond.

The term "substituted", as used herein, refers to an atom that has one or more substitutions or one or more substituents that may be the same or different and may include a hydrogen substituent. Thus, the terms alkyl, cycloalkyl, alkenyl, alkoxy, alkanoyl, and aryl, for example, refer to groups that may include substituted variations. Substituted variations include linear, branched, and cyclic variations, and groups that have a substituent or substituents that replace one or more hydrogens attached to any carbon atom in the group.

In general, a compound can contain one or more chiral centers. Compounds containing one or more chiral centers can include those described as an "isomer", a "stereoisomer", a "diastereomer", an "enantiomer", an "optical isomer" or as a "racemic mixture". Conventions for stereochemical nomenclature, eg, the Cahn, Ingold, and Prelog rules for stereoisomer nomenclature, as well as methods for the determination of stereochemistry and stereoisomer separation, are known in the art. See, for example, Michael B. Smith and Jerry March, March's Advanced Organic Chemistry, 5th Edition, 2001. The compounds and structures of this disclosure are intended to encompass all possible optical isomers, stereoisomers, diastereomers, enantiomers, and / or isomers. which would be understood to exist for the specified compound or structure, including any mixture, racemic or otherwise, thereof.

This disclosure encompasses any and all tautomeric forms, solvated or unsolvated, hydrated or non-hydrated, as well as any isotopic form of atoms of the compounds and compositions disclosed herein.

This disclosure encompasses any and all crystalline polymorphs or different crystalline forms of the compounds and compositions disclosed herein.

Example protocol for determining attenuation in vitro

One day prior to transfection, cells were plated in a 96-well plate at 2 x 103 cells per well with 100 µl DMEM (HyClone cat # SH30243.01) containing 10% FBS. % and were grown in an incubator at 37 ° C containing a humidified atmosphere of 5% CO ² in air. Prior to transfection, the medium was changed to 90 µl Opti-MEM I Reduced Serum Medium (Life Technologies Cat # 31985-070) containing 2% FBS. Then, 0.2 µl of Lipofectamine RNAiMax (Cat # 13778-100 from Life Technologies) was mixed with 4.8 µl of Opti-MEM I for 5 minutes at room temperature. Next, 1 µl siRNA was mixed with 4 µl Opti-MEM I and combined with the LF2000 solution, and mixed gently, without vortexing. After 5 minutes at room temperature, the mixture was incubated for an additional 10 minutes at room temperature to allow the RNA-RNAiMax complexes to form. In addition, 10 µl of RNA-RNAiMax complexes were added to a well and the plate was gently shaken by hand. Cells were incubated in a 37 ° C incubator containing a humidified atmosphere of 5% CO ² in air for 2 hours. The medium was exchanged for fresh Opti-MEM I reduced serum medium containing 2% FBS. 24 hours after transfection, cells were washed once with ice-cold PBS. Cells were lysed with 50 µl of Cellto-Ct lysis buffer (Cat # 4391851 C from Life Technologies) for 5-30 minutes at room temperature. 5 µl of stop solution was added and incubated for 2 minutes at room temperature. The mRNA level was measured by RT-qPCR with TAQMAN immediately. Samples can be frozen at -80 ° C and tested later.

Example protocol for determining stability in serum

0.2 mg / ml siRNA was incubated with 10% human serum at 37 ° C. At certain time points (0, 5, 15 and 30 min), aliquots of 200 µl of sample were taken and extracted with 200 µl of extraction solvent (chloroform: phenol: isoamyl alcohol = 24: 25: 1) . The sample was vortexed and centrifuged at 13,000 rpm for 10 min at RT, then the upper phase solution was transferred and filtered with a 0.45 µm filter. The filtrate was transferred to a 300 µl HPLC injection vial. For LC-MS, the mobile phase was MPA: 100 mM HFIP 7 mM TEA in H ² O, MPB: 50% methanol, 50% acetonitrile. The column: Waters Acquity OST 2.1 x 50 mm, 1.7 | im.

Examples

Example 1: The siRNA formulation of GST-rc and p21 of this invention showed profound reduction in cancer xenograft tumors in vivo. The GST-rc and p21 siRNAs provided gene attenuation potency in vivo when administered in a liposomal formulation to cancer xenograft tumors.

Figure 1 shows the tumor inhibition efficacy for a liposomal siRNA formulation of GST-rc (SEQ ID NO: 61 and 126) and p21 (SEQ ID NO: 341 and 355, N = U). A cancer xenograft model was used with a relatively low dose of 1.15 mg / kg for GST-rc siRNA and 0.74 mg / kg for p21 siRNA.

The siRNA formulation of GST-rc and p21 showed significant and unexpectedly advantageous tumor inhibition efficacy within a few days after administration. After 30 days, the siRNAs of GST-rc and p21 showed a remarkably advantageous tumor inhibition efficiency, with a tumor volume reduced by more than 2 times compared to the control.

GST-rc siRNA was administered in four injections (day 1, 8, 15 and 22) of a liposomal formulation having the composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25:30: 20:20: 5).

For the cancer xenograft model, a A549 cell line was obtained from ATCC. Cells were kept in Culture medium supplemented with 10% fetal bovine serum and 100 U / ml penicillin and 100 µg / ml streptomycin. The cells were divided 48 hours before inoculation so that the cells were in logarithmic growth phase when harvested. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and determined on a hemocytometer in the presence of trypan blue (only viable cells are counted). Cells were resuspended at a concentration of 5 * 107 / ml in serum-free medium. Then, the cell suspension was mixed well with ice-thawed BD Matrigel in a 1: 1 ratio for injection.

The mice were nude nude (nu / nu) female mice from the Charles River Laboratory, immunosuppressed, 6 to 8 weeks old, 7 to 8 mice per group.

For tumor model preparation, each mouse was inoculated subcutaneously on the right flank with 0.1 ml of a 2.5 * 106 inoculum of A549 cells using a 25 G needle and syringe, one inoculum per mouse. The mice were not anesthetized for inoculation.

For tumor volume measurements and randomization, tumor size was measured to the nearest 0.1 mm. Tumor volumes were calculated using the formula: tumor volume = length x width2 / 2. Once the established tumors reached approximately 120-175 mm3, the average tumor volume was approximately 150 mm3, the mice were assigned to the various vehicle and treatment control groups so that the mean volumes of the tumors in the Treated groups were within 10% of the mean tumor volume in the vehicle control group, ideally, the% CV of the tumor volume was less than 25%. On the same day, the test articles and the control vehicle were administered according to the dosage schedule. Tumor volumes were monitored three times during week 1, twice during the rest of the weeks, including the day of study completion.

For dosing administration, on the day of dosing, the test items were removed from the -80 ° C freezer and thawed on ice. Before applying it to the syringes, the bottle containing the formulation was inverted with the hands a few times. All test articles were dosed by i.v., at 10 ml / kg.

To determine body weight, mice were weighed with a precision of 0.1 g. Body weights were monitored and recorded daily within 7 days after the first dose was administered. Body weights were monitored and recorded twice a week, for the remainder of the weeks, including the day of study completion.

For tumor harvesting, 28 days after the first dose, tumor volume was measured and the tumor was dissected for weight measurement and stored for PD biomarker study. The weight of the tumor was recorded.

Example 2: The siRNA formulation of GST-rc and p21 of this invention showed profound reduction in cancer xenograft tumors in vivo. The GST-rc and p21 siRNAs provided gene attenuation potency in vivo when administered in a liposomal formulation to cancer xenograft tumors.

Figure 2 shows the tumor inhibition efficacy for a liposomal formulation of siRNA of GST-rc (SEQ ID N °: 156 and 182) and p21 (SEQ ID N °: 341 and 355, N = U). A cancer xenograft model was used with a relatively low dose of 0.75 mg / kg for each siRNA.

The siRNA formulation of GST-rc and p21 showed significant and unexpectedly advantageous tumor inhibition efficacy within a few days after administration. After 30 days, the GST-rc and p21 siRNAs showed remarkably advantageous tumor inhibition efficacy, with a 1.7-fold reduced tumor volume compared to the control.

Example 3: The siRNA formulations of GST-rc and p21 of this invention demonstrated increased cancer cell death by apoptosis of cancer cells in vitro. Cancer cell apoptosis was monitored in vitro by observing the up-regulation of PUMA, a biomarker for apoptosis, which is associated with loss of cell viability.

The siRNA formulations of GST-rc and p21 provided an unexpected increase in apoptosis of cancer cells.

As shown in Figure 3, the expression level of PUMA for a siRNA formulation of GST-rc and p21 (Figure 3, p21 (A) GSTP (A), SEQ ID NO: 341 and 355 of P21, N = U, GSTP SEQ ID NO: 156 and 182) was greatly increased from about 2-4 days after transfection of the GST-rc and p21 siRNAs.

These data show that the siRNA formulations of GST-rc and p21 of this invention provided an unexpected increase in apoptosis of cancer cells.

The protocol for the PUMA biomarker was as follows. One day prior to transfection, cells were plated in a 96-well plate at 2 x 103 cells per well with 100 µl DMEM (HyClone cat # SH30243.01) containing 10% FBS. % and were grown in an incubator at 37 ° C containing a humidified atmosphere of 5% CO ² in air. The following day, prior to transfection, the medium was replaced with 90 µl of Opti-MEM I Reduced Serum Medium (Life Technologies Cat # 31985-070) containing 2% FBS. Then, 0.2 µl of Lipofectamine RNAiMax (Cat # 13778-100 from Life Technologies) was mixed with 4.8 µl of Opti-MEM I for 5 minutes at room temperature. 1 µl of siRNAs (1 µM stock conc.) Was mixed with 4 µl of Opti-MEM I and combined with the RNAiMAX solution, then mixed gently. The mixture was incubated for 10 minutes at room temperature to allow the RNA-RNAiMAX complexes to form. 10 µl of RNA-RNAiMax complexes were added per well, to a final concentration of 10 nM. Cells were incubated for 2 hours and the medium was exchanged for fresh Opti-MEM I reduced serum medium containing 2% FBS. 1, 2, 3, 4, and 6 days after transfection, cells were washed once with ice-cold PBS and then lysed with 50 µl Cell-to-Ct Lysis Buffer (Cat # 4391851 C from Life Technologies) for 5-30 minutes at room temperature. 5 µl of stop solution was added and incubated for 2 minutes at room temperature. PUMA (BBC3, Cat # Hs00248075, Life Technologies) mRNA levels were measured by qPCR with TAQMAN.

Example 4: An in vitro double attenuation study of siRNAs targeting GST-n and p21 showed that the combination was very active in suppressing the mRNA levels of both proteins. As shown in Table 11, the level of attenuation for each of GST-n and p21 was high for concentrations of 2, 10 and 50 nM.

Table 11: double attenuation on A549

Protocol: transfect with siRNA of P21 at 2, 10 and 50 nM. After 1,2,3 or 4 days, transfect with GST-n siRNA at 10 nM. 1 day later, analyze by RT-PCR to determine the levels of P21 mRNA and GST-K mRNA.

Example 5: A549 orthotopic lung cancer mouse model. The GST-n siRNAs comprised in the compositions of this invention, as defined in the claims, may exhibit profound reduction in orthotopic lung cancer tumors in vivo. In this example, a GST-n siRNA provided gene attenuation potency in vivo when administered in a liposomal formulation to orthotopic lung cancer tumors in nude nude mice.

In general, an orthotopic tumor model can have direct clinical relevance to the efficacy and potency of a drug, as well as improved predictive ability. In the orthotopic tumor model, tumor cells implant directly into the same kind of organ from which the cells originated.

The antitumor efficacy of the siRNA formulation against A549 human lung cancer was evaluated by comparing the final weights of primary tumors measured at necropsy for the treatment group and the vehicle control group.

Figure 4 shows the inhibition of an orthotopic lung cancer tumor in vivo for a GST-n siRNA based on the BU2 structure (SEQ ID NO: 61 and 126). An A549 orthotopic lung cancer mouse model was used with a relatively low dose of 2 mg / kg of the siRNA targeting GST-n.

The GST-n siRNA showed significant and unexpectedly advantageous lung tumor inhibition efficacy in this six week study. As shown in Figure 4, after 43 days, the GST-n siRNA showed remarkably advantageous tumor inhibition efficacy, with the average final tumor weights significantly reduced by 2.8-fold compared to the control. .

For this study, male NCr nu / nu mice, 5-6 weeks old, were used. The experimental animals were kept in a HEPA filtered environment during the experimental period. SiRNA formulations were stored at 4 ° C prior to use and warmed to room temperature 10 minutes prior to injection into the mouse.

For this A549 human orthotopic lung cancer model, on the day of surgical orthotopic implantation (SOI), stem tumors were harvested from the subcutaneous site of animals bearing an A549 tumor xenograft and placed in RPMI-1640 medium. Necrotic tissues were extracted and viable tissues were cut into 1.5-2 mm3 pieces. Animals were anesthetized with isoflurane inhalation and the surgical area was sterilized with iodine and alcohol. A transverse incision approximately 1.5 cm in length was made on the left side of the mouse chest wall using a pair of surgical scissors. An intercostal incision was made between the third and fourth rib and the left lung was exposed. An A549 tumor fragment was transplanted to the surface of the lung with an 8-0 surgical suture (nylon). The chest wall was closed with a 6-0 surgical suture (silk). The lung was re-inflated by intrathoracic puncture using a 3 cc syringe with a 25 G X 1 A needle to remove the remaining air in the chest cavity. The chest wall was closed with a 6-0 silk surgical suture. All procedures of the operation described above were performed under a microscope at 7x magnification under HEPA filtered laminar flow hoods.

Three days after tumor implantation, the tumor-bearing model mice were randomly divided into groups of ten mice per group. For the group of interest, treatment of the ten mice was started three days after tumor implantation.

For the interest group, the formulation was (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K: DSPE-PEG-2K), a liposomal composition. The liposomes encapsulated the GST-rc siRNA.

By the end of the study, the test mice were sacrificed forty-two days after the start of treatment. Primary tumors were excised and weighed on an electronic balance for further analysis.

For an estimate of the toxicity of the compound, the mean body weight of the mice in the treated and control groups remained within the normal range throughout the experimental period. No other symptoms of toxicity were observed in the mice.

Example 6: the GST-rc siRNAs comprised in the compositions of this invention, as defined in the claims, showed profound reduction in cancer xenograft tumors in vivo. GST-rc siRNAs provided gene attenuation potency in vivo when administered in a liposomal formulation to cancer xenograft tumors.

Figure 5 shows the tumor inhibition efficiency for a GST-rc siRNA (SEQ ID NO: 156 and 182). A cancer xenograft model was used with a relatively low dose of 0.75 mg / kg siRNA targeting GST-rc.

GST-rc siRNA showed significant and unexpectedly advantageous tumor inhibition efficacy within a few days of administration. After 36 days, the rc-GST siRNA showed remarkably advantageous tumor inhibition efficacy, with a tumor volume doubled compared to the control.

As shown in Figure 5, the GST-rc siRNA demonstrated significant and unexpectedly advantageous tumor inhibition efficiency on the final day. In particular, the weight of the tumor more than doubled.

The GST-rc siRNA was administered in two injections (day 1 and 15) of a liposomal formulation having the composition (ionizable lipid: cholesterol: DOPE: DoPC: DPPE-PEG-2K) (25: 30: 20: 20: 5).

For the cancer xenograft model, a A549 cell line was obtained from ATCC. The cells were maintained in culture medium supplemented with 10% fetal calf serum and 100 U / ml penicillin and 100 µg / ml streptomycin. The cells were divided 48 hours before inoculation so that the cells were in logarithmic growth phase when harvested. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and determined on a hemocytometer in the presence of trypan blue (only viable cells are counted). Cells were resuspended at a concentration of 5 * 107 / ml in serum-free medium. Then, the cell suspension was mixed well with ice-thawed BD Matrigel in a 1: 1 ratio for injection.

The mice were nude nude (nu / nu) female mice from the Charles River Laboratory, immunosuppressed, 6 to 8 weeks old, 7 to 8 mice per group.

For tumor model preparation, each mouse was inoculated subcutaneously on the right flank with 0.1 ml of a 2.5 * 106 inoculum of A549 cells using a 25 G needle and syringe, one inoculum per mouse. The mice were not anesthetized for inoculation.

For tumor volume measurements and randomization, tumor size was measured to the nearest 0.1 mm. Tumor volumes were calculated using the formula: tumor volume = length x width2 / 2. Once established tumors reached approximately 120-175 mm3, the average tumor volume was approximately 150 mm3, mice were assigned to the various vehicle and treatment control groups so that the mean volumes of tumors in the treated groups were within 10% of the mean tumor volume in the vehicle control group, ideally, the% CV of the tumor volume was less than 25%. On the same day, the test articles and the control vehicle were administered according to the dosage schedule. Tumor volumes were monitored three times during week 1, twice during the rest of the weeks, including the day of study completion.

For dosing administration, on the day of dosing, the test items were removed from the -80 ° C freezer and thawed on ice. Before applying it to the syringes, the bottle containing the formulation was inverted with the hands a few times. All test articles were dosed at 0.75 mg / kg per i.v., every two weeks X 2, at 10 ml / kg.

To determine body weight, mice were weighed with a precision of 0.1 g. Body weights were monitored and recorded daily within 7 days after the first dose was administered. Body weights were monitored and recorded twice a week, for the remainder of the weeks, including the day of study completion.

For tumor harvesting, 28 days after the first dose, tumor volume was measured and the tumor was dissected for weight measurement and stored for PD biomarker study. The weight of the tumor was recorded.

Example 7: GST-rc siRNAs comprised in the compositions of this invention, as defined in the claims, demonstrated increased cancer cell death by cancer cell apoptosis in vitro. The GST-rc siRNAs provided attenuation of GST-rc, resulting in up-regulation of PUMA, a biomarker for apoptosis and associated with loss of cell viability.

The GST-rc siRNA, SEQ ID NO: 156 and 182, which contained a combination of deoxynucleotides in the seed region, a 2'-F substituted deoxynucleotide and 2'-OMe substituted ribonucleotides, provided an unexpected increase in apoptosis of cancer cells.

The expression level of PUMA for GST-rc siRNA, SEQ ID NO: 156 and 182, was measured as shown in Figure 6. In Figure 6, PUMA expression increased considerably from 2-4 days after GST-rc siRNA transfection.

These data show that the structure of GST-rc siRNAs containing a combination of deoxynucleotides in the seed region, a 2'-F substituted deoxynucleotide and 2'-OMe substituted ribonucleotides, provided an unexpected increase in apoptosis of cells. cancerous

The protocol for the PUMA biomarker was as follows. One day prior to transfection, cells were plated in a 96-well plate at 2 x 103 cells per well with 100 µl DMEM (HyClone cat # SH30243.01) containing 10% FBS. % and were grown in an incubator at 37 ° C containing a humidified atmosphere of 5% CO ² in air. The following day, prior to transfection, the medium was replaced with 90 µl of Opti-MEM I Reduced Serum Medium (Life Technologies Cat # 31985-070) containing 2% FBS. Then, 0.2 µl of Lipofectamine RNAiMax (Cat # 13778-100 from Life Technologies) was mixed with 4.8 µl of Opti-MEM I for 5 minutes at room temperature. 1 µl of the GST-rc siRNA (1 µM stock conc.) Was mixed with 4 µl of Opti-MEM I and combined with the RNAiMAX solution, and mixed gently. The mixture was incubated for 10 minutes at room temperature to allow the RNA-RNAiMAX complexes to form. 10 µl of RNA-RNAiMax complexes were added per well, to a final siRNA concentration of 10 nM. Cells were incubated for 2 hours and the medium was exchanged for fresh Opti-MEM I reduced serum medium containing 2% FBS. 1, 2, 3, 4, and 6 days after transfection, cells were washed once with ice-cold PBS and then lysed with 50 µl Cell-to-Ct Lysis Buffer (Cat # 4391851 C from Life Technologies) for 5-30 minutes at room temperature. 5 µl of stop solution was added and incubated for 2 minutes at room temperature. PUMA (BBC3, Cat # Hs00248075, Life Technologies) mRNA levels were measured by qPCR with TAQMAN.

Example 8: The rc-GST siRNAs comprised in the compositions of this invention, as defined in the claims, may exhibit profound shrinkage of cancer xenograft tumors in vivo. GST-rc siRNAs can provide gene attenuation potency in vivo when administered in a liposomal formulation to cancer xenograft tumors.

Figure 7 shows the tumor inhibition efficiency for a GST-rc siRNA (SEQ ID NO: 61 and 126). Dose-dependent attenuation of GST-rc mRNA was observed in vivo with siRNA targeting GST-rc. A cancer xenograft model with a GST-rc targeting siRNA was used.

GST-rc siRNA showed significant and unexpectedly advantageous tumor inhibition efficacy within a few days of administration. As shown in Figure 7, treatment with a GST-rc siRNA resulted in a significant reduction in GST-rc mRNA expression 4 days after injection into a lipid formulation. At the highest dose of 4 mg / kg, a significant reduction of approximately 40% was detected 24 hours after injection.

The p21 siRNA was administered in a single 10 ml / kg injection of a liposomal formulation having the composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25: 30: 20: 20: 5) .

For the cancer xenograft model, a A549 cell line was obtained from ATCC. The cells were maintained in culture medium supplemented with 10% fetal calf serum and 100 U / ml penicillin and 100 µg / ml streptomycin. The cells were divided 48 hours before inoculation so that the cells were in logarithmic growth phase when harvested. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and determined on a hemocytometer in the presence of trypan blue (only viable cells are counted). Cells were resuspended at a concentration of 4 * 107 / ml in serum-free medium. Then, the cell suspension was mixed well with ice-thawed BD Matrigel in a 1: 1 ratio for injection.

The mice were nude nude (nu / nu) female mice from the Charles River Laboratory, immunosuppressed, 6 to 8 weeks old, 3 mice per group.

For tumor model preparation, each mouse was inoculated subcutaneously on the right flank with 0.1 ml of a 2.5 * 106 inoculum of A549 cells using a 25 G needle and syringe, one inoculum per mouse. The mice were not anesthetized for inoculation.

For tumor volume measurements and randomization, tumor size was measured to the nearest 0.1 mm. Tumor volumes were calculated using the formula: tumor volume = length x width2 / 2. Tumor volumes were monitored twice weekly. Once established tumors reached approximately 350-600 mm3, mice were assigned to groups with varying time points. On the same day, the test articles were administered according to the dosage regimen.

For dosing administration, on the day that established tumors reached approximately 350-600 mm3, the test articles were removed from the refrigerator at 4 ° C. Before applying it to the syringes, the bottle containing the formulation was inverted with the hands a few times to homogenize the solution.

To determine body weight, mice were weighed with a precision of 0.1 g. Body weights were monitored and recorded twice a week, for the remainder of the weeks, including the day of study completion.

For tumor harvesting, animals were sacrificed by CO ² overdose and tumors were dissected at 0, 24, 48, 72, 96 (optional) and 168 hours after dosing. Tumors were first weighed wet and then separated into three parts for KD, distribution and biomarker analysis. Samples were snap frozen in liquid nitrogen and stored at -80 ° C until ready to be processed.

The embodiments and aspects of the disclosure described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation to identify nucleic acid molecules with enhanced iRNA activity.

It is understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing only particular embodiments. It will be readily apparent to one skilled in the art that various substitutions and modifications can be made to the description disclosed herein without departing from the scope and spirit of the description, and that those embodiments are within the scope of this description and the appended claims.

It should be noted that, as used herein and in the appended claims, the singular forms "a / an", "an" and "the" include the plural reference unless the context clearly indicates otherwise. . Furthermore, the terms "a / one" (or "an"), "one or more" and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprises", "comprising", "containing", "including" and "having" can be used interchangeably, and will be read broadly and without limitation.

The enumeration of ranges of values in this document is intended simply to serve as a shorthand method for individually referring to each separate value that falls within the range, unless otherwise noted herein, and each value separately it is incorporated in the specification as if individually listed herein. For Markush groups, those skilled in the art will recognize that this description includes individual members as well as subgroups of Markush group members.

Without further elaboration, it is believed that one skilled in the art can utilize, based on the foregoing description, the present invention to its fullest extent.

All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.

Claims (15)

1. Composition comprising iRNA molecules targeting a human rc-GST, iRNA molecules targeting a human p21 encoded by a CDKN1A gene, and a pharmaceutically acceptable carrier. Composition according to claim 1, in which: I) each of the GST-rc targeting iRNA molecules has an antisense strand SEQ ID NO: 131 and a sense strand SEQ ID NO: 157; II) each of the iRNA molecules targeting p21 has an antisense strand SEQ ID NO: 341 and a sense strand SEQ ID NO: 355; III) each of the GST-rc targeting iRNA molecules has an antisense strand SEQ ID NO: 156 and a sense strand SEQ ID NO: 182; or IV) Each of the p21 targeting iRNA molecules has an antisense strand SEQ ID NO: 343 and a sense strand SEQ ID NO: 357. Composition according to claim 1, in which one or more of the nucleotides in the duplex region of the iRNA molecules are chemically modified or modified, preferably in which the chemically modified or modified nucleotides are 2'-deoxynucleotides, substituted nucleotides with 2'-O-alkyl, 2'-deoxy-2'-fluoro substituted nucleotides, phosphorothioate nucleotides, blocked nucleotides, or any combination thereof. Composition according to claim 1, in which each of the iRNA molecules contains a deoxynucleotide T at one or more of positions 2 to 8 from the 5 'end of the antisense strand. Composition according to claim 1, in which the antisense chain of each of the iRNA molecules has deoxynucleotides in a plurality of positions, the plurality of positions being one of the following: each of positions 4, 6 and 8, from the 5 'end of the antisense strand; each of positions 3, 5 and 7, from the 5 'end of the antisense chain; each of positions 1, 3, 5 and 7, from the 5 'end of the antisense chain; each of positions 3-8, from the 5 'end of the antisense chain; or each of positions 5-8, from the 5 'end of the antisense strand. Composition according to claim 1, wherein the composition inhibits the expression of GST-rc mRNA with an IC50 of less than 50 pM, and inhibits the expression of p21 mRNA with an IC50 of less than 50 pM, or in where a single administration of the composition inhibits the expression of GST-rc mRNA levels in vivo by at least 25%. 7. Composition according to claim 1, wherein the carrier comprises: I) liposomal nanoparticles that encapsulate the iRNA molecules; II) liposomal nanoparticles that encapsulate the iRNA molecules and retain at least 80% of the encapsulated iRNA molecules after 1 hour of exposure to human serum; or III) Liposomal nanoparticles having a size of 10 to 1000 nm, or 10 to 150 nm. Composition according to claim 1, in which the composition is active to treat a malignant tumor, preferably in which the malignant tumor is located in the lung, colon, kidney, pancreas, liver, bone, skin or intestine. Composition according to claim 1, in which the carrier comprises liposomal nanoparticles comprising an ionizable lipid, a structural lipid, one or more stabilizing lipids, and a lipid to reduce the immunogenicity of the nanoparticles, preferably in which the ionizable lipid is selected from the group of the following compounds:

Composition according to any one of claims 1-9, for use in a method of preventing, treating or ameliorating one or more symptoms of a malignant tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of said composition. Composition according to claim 10 for use according to claim 10, wherein: I) the malignant tumor is associated with the KRAS mutation, the method further comprising identifying a tumor cell in the subject, the tumor cell comprising at least one of: (i) a KRAS gene mutation, and (ii) a level of aberrant expression of the KRAS protein II) the malignant tumor overexpress GST-tc; or III) iRNA molecules decrease the expression of GST-tc and p21 in the subject. Composition according to claim 10 for use according to claim 10, wherein in the method the administration: I) decrease the expression of GST-tc and p21 in the subject by at least 5% for at least 5 days; II) decreases the volume of the malignant tumor in the subject by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40% or at least 50 %; III) reduces the growth of malignant tumor cells in the subject; or IV) reduces growth for at least 2%, or at least 5%, or at least 10%, or at least 15% or at least 20% of the malignant tumor cells in the subject. A composition according to claim 10 for use according to claim 10, wherein the method reduces one or more symptoms of the malignant tumor, or delays or terminates the progression of the malignant tumor. Composition according to claim 10 for use according to claim 10, wherein the malignant tumor is colon cancer, pancreatic cancer, kidney cancer, lung cancer, breast cancer or fibrosarcoma, or wherein the tumor malignant is lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas or colorectal carcinoma. Composition according to claim 10 for use according to claim 10, wherein in the method the administration: I) is carried out from 1 to 12 times per day; II) is carried out for a duration of 1,2, 3, 4, 5, 6 or 7 days; III) is carried out for a duration of 1,2, 3, 4, 5, 6, 8, 10 or 12 weeks; IV) is a dose of from 0.01 to 2 mg / kg of the iRNA molecules at least once per day for a period of up to twelve weeks; V) provides a mean AUC (O-ultimate) of from 1 to 1000 ug * min / ml and a mean Cmax of from 0.1 to 50 ug / ml for the GST-rc iRNA molecule; VI) provides a mean AUC (O-ultimate) of from 1 to 1000 ug * min / ml and a mean Cmax of from 0.1 to 50 ug / ml for the p21 iRNA molecule; or VII) is an intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, oral, topical, infusion or inhalation.